Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of <Emphasis Type="Italic">Saussurea medusa</Emphasis>

Three previously established cell lines (yellow, red and white) of Saussurea medusa were investigated for jaceosidin and hispidulin production. Maximum yields of the jaceosidin and hispidulin were obtained in the red cell line at 75 ± 0.41 and 6.4 ± 0.31 mg l^-1. Production of jaceosidin and hispidulin correlated with the sizes of compact callus aggregates (CCA) and cellular viability. In the red cell line, the sizes of CCA were predominantly of 2–4mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.Revisions requested 3 September 2004/27 October 2004; Revisions received 22 October 2004/18 November 2004     

Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa

Calli cultures derived from the leaves of Saussurea medusa were selected on the basis of colour into three callus, A, B and C, which suggested different levels of metabolite accumulation. An improved reversed phase high performance liquid chromatographic method provided selective determination of the jaceosidin content of these samples. The jaceosidin concentration in callus B was higher than that of the callus A and C. By using 12-day old culture and 9-day old inoculum, jaceosidin yield of 72.91 mg l^–1was obtained from cell line B in cell suspension cultures. The influence of some factors affecting jaceosidin formation, i.e. temperature, light, inoculum size, type of media, phytohormones, nitrogen and carbon source etc. were also examined. Light irradiation and combination of 3% (w/v) sucrose with 1% glucose brought about a marked increase of jaceosidin production. The effect of blue light on jaceosidin was markedly superior to other kinds of monochromatic light (red and far-red) or white light. Analysis of growth and jaceosidin content of callus cultures and cell suspension cultures demonstrated that the production of jaceosidin was growth-dependent in both cell solid culture and cell suspension culture.     

Comparison of Techniques for the Extraction of Flavonoids from Cultured Cells of Saussurea medusa Maxim

Summary Room temperature, heat reflux, Soxhlet, ultrasound-assisted and microwave-assisted extractions were employed to extract flavonoids from dry cell cultures of Saussurea medusa Maxim. 24, 20, 6, 0.5 and 0.1 h exposures were necessary for room temperature, Soxhlet, heat reflux, ultrasound-assisted and microwave-assisted extractions to obtain the maximum yield of flavonoids of 3.0%, 4.1%, 3.9%, 3.5% and 4.1%, respectively. Microwave-assisted extraction showed obvious advantages in short duration and high efficiency to extract flavonoids from S. medusa cultured cells.     

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in <Emphasis Type="Italic">Saussurea medusa</Emphasis>

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate     

Spectral composition of irradiation regulates the cell growth and flavonoids biosynthesis in callus cultures of Saussurea medusa Maxim

Cell growth, flavonoids biosynthesis and L-phenylalanine ammonia-lyase (PAL) activity were studied in callus cultures of Saussurea medusa Maxim. under different types of spectral radiance. After 21 days, red light significantly improved the callus growth, but inhibited the biosynthesis of flavonoids in callus cultures. However, blue light was found to enhance the biosynthesis of flavonoids, although callus growth under this spectrum was comparable with that under white and other coloured spectra, such as green and yellow. The accumulation of flavonoids in callus cultures was related to the PAL activity, which was found to be stimulated by the spectral composition of irradiation.     

Comparison of Differences in Chemical Composition and Related Antioxidant Activity of Snow Lotus from Different Origins

The snow lotus is an endangered traditional Chinese medicinal herb. Saussurea involucrata, Saussurea laniceps, and Saussurea medusa, the three main snow lotus species (five herbs and two S. involucrata cell cultures), were selected for this study. Snow lotus (XLs) was extracted using 75 % (v/v) ethanol. Two reversed phase-high performance liquid chromatography-diode array detector methods were developed and validated for the determination of 10 representative components in XLs. The antioxidant efficacy of XLs and their components was investigated using DPPH, ABTS free radical scavenging, and ROS inhibition experiments. The results showed that the IC₅₀ for DPPH scavenging ranged from 0.06–0.29 mg/mL for XLs and from 0.13–0.63 mg/mL for ABTS, and could downregulate ROS to varying degrees. The results of the antioxidant activity showed that rutin, quercetin, and isochlorogenic acid A contributed to the antioxidant capacity of XLs. The high content and activity of the cell cultures indicate that they can serve as an effective alternative to snow lotus, thus providing a theoretical basis for the selection of herbs and cell cultures to fulfil various needs.     

Quality evaluation of snow lotus (Saussurea): quantitative chemical analysis and antioxidant activity assessment

Snow lotus is commonly used as a medicinal plant and has great pharmacological value. To protect these endangered plants, in vitro propagation and cell cultures have been established in order to meet the growing market demand. The phenolic composition, antioxidant activities, total phenolic content (TPC) and total flavonoid content (TFC) from three most commonly used species, in vitro propagated lines and the cell cultures were investigated to qualify their pharmacological value. Quantitative analysis showed that the phenolics varied greatly among different species and the same species at different habitats. From this it can be inferred that the phenolics were influenced by genetic background and the environmental conditions. Significant correlations were observed between the antioxidant activity and several phenolics/TPC/TFC, suggesting that the phenolics are a major contributor of the antioxidant activity and are important for quality evaluation of snow lotus. Based on the abundance of phenolics, TPC, TFC and antioxidant activity, the order of the quality for wild species would be Saussurea involucrata > Saussurea medusa > Saussurea gossypiphora. For S. medusa, its quality judged by origin would be Shigatse > Lhasa > Nagqu. For in vitro propagated plants, the matured plants could be a reliable substitute for wild plants, and the dynamics of phenolics is critical for quality control of this monocarpic species. We provide the first report of quality comparison between the wild plants and the cell cultures. The advantages of developing cell cultures as alternatives for plants collected from the wild are discussed.     

Improved cell growth and total flavonoids of Saussurea medusa on solid culture medium supplemented with rare earth elements

Callus cultures of Saussurea medusa were cultivated on solid culture medium supplemented with either Ce³⁺, La³⁺, Nd³⁺ or a mixture of rare earth elements. Ce³⁺, 0.05 mM, gave the highest biomass (0.53 g dry wt per flask) and total flavonoids (27.5 mg per flask), which were, 70% and 100% higher than those without Ce³⁺ addition, respectively. Ce³⁺, 0.01–0.1 mM, or La³⁺, 0.05 mM, or the mixture of rare earth elements, 0.025–0.1 mM, can substitute for 6-benzyladenine, and 0.025 mM Ce³⁺ can partly substitute for naphthaleneacetic acid in promoting cell growth and biosynthesis of total flavonoids in S. medusa.     

水母雪莲通气组织形成的相关基因SmLSD1克隆及表达

该研究以水母雪莲为实验材料,通过RT-PCR结合RACE技术克隆了通气组织形成相关基因SmLSD1(GenBank登录号为OL690334),并对该基因在不同胁迫下的表达量及编码蛋白结构进行测定分析。结果表明：(1)水母雪莲SmLSD1基因全长965 bp,包含537 bp的开放阅读框,编码178个氨基酸。(2)同源序列比对发现,水母雪莲SmLSD1蛋白与菊科植物牛蒡LSD1的氨基酸序列相似性最高,达到98.31%。(3)亚细胞定位显示SmLSD1基因主要在细胞核和细胞膜上表达;原核表达显示,SmLSD1基因编码氨基酸的分子量约为18 kD。(4)荧光定量分析显示,SmLSD1基因在根、茎、叶中均有表达,且在叶片中表达量最高;在低温、低氧及紫外胁迫下,SmLSD1基因的表达量下调。研究推测,SmLSD1基因在水母雪莲通气组织的形成以及对逆境胁迫的响应中发挥着重要作用。     

Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.     

Salicylic Acid Enhances Jaceosidin and Syringin Production in Cell Cultures of Saussurea medusa

Addition of 20 μM salicylic acid to Saussurea medusa cell cultures at day 6 resulted in jaceosidin and syringin productions up to 95 mg l⁻¹ and 631 mg l⁻¹ which were, respectively, about 2.5- and 2.7-fold higher than in the control. The biomass was increased from 8 to 12 g l⁻¹. Expression of chalcone synthase gene (chs) increased sharply after 12 h treatment and was sustained up to 48 h; chalcone isomerase gene (chi) expression reached a peak at 24 h and decreased after 48 h; and phenylalanine ammonia-lyase activity increased by 7.5-fold (96 U mg⁻¹ protein) higher than in the control after 24 h. These results indicate that salicylic acid enhances the production of jaceosidin and syringin which is accompanied by induction of the related phenylpropanoid biosynthetic enzymes.     

Syringin production by <Emphasis Type="Italic">Saussurea medusa</Emphasis> cell cultures in a novel bioreactor

The culture of Saussurea medusa cell were cultured in an internal loop airlift bioreactor with sifter draft tube (ILABSDT) was investigated. Under the optimal culture conditions, which were inoculation size 1.5 g(d.m.) dm⁻³, aeration rate 0.3 dm³(air) dm⁻³(medium) min⁻¹, and 14 mesh sifter holes, the maximum biomass, syringin content and syringin production reached 11.7 g(d.m.) dm⁻³, 17.7 mg g⁻¹ and 206.6 mg dm⁻³, respectively. Among cell cultures in shake flask, bubble column bioreactor and ILABSDT, ILABSDT had the highest syringin productivity and reached 12.41 mg dm⁻³ d⁻¹.     

Engineering of red cells of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and comparative genome-wide gene expression analysis of red cells versus wild-type cells

We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC–MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1–GL3/TT8–PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.     

Identification of a cell wall peroxidase in red calli of Prunus incisa Thunb.

Red calli occur frequently in callus cultures of Prunus incisa Thunb. Calli that become red in color stop growing and turn brown while calli that remain green continue to grow. This study was carried out to compare the accumulation of antioxidant activity in red and green calli. The anthocyanin content, peroxidase isoforms and peroxidase activity were different in red and green calli. Red calli contained higher levels of anthocyanins, cell wall peroxidase activity and lower soluble peroxidase activity than green calli. A basic cell wall peroxidase (pI 10.0) was present only in red calli. Two acidic peroxidases (pI 6.0 and 6.8) had higher accumulation in green calli than in red calli. In the cell wall fraction of red calli, a peroxidase isoform with an apparent molecular mass of 30 kDa was found. MALDI -TOF mass spectrometry and internal amino acid sequence analysis indicate that this protein has a very high similarity with the cell wall peroxidase of Beta vulgarisL .     

The effects of pollen limitation on population dynamics of snow lotus (<Emphasis Type="Italic">Saussurea medusa</Emphasis> and <Emphasis Type="Italic">S. laniceps</Emphasis>, Asteraceae): Threatened Tibetan medicinal plants of the eastern Himalayas

Pollen limitation reduces seed production and may reduce plant population growth rate. Plants may be particularly prone to pollen limitation if they require pollinators, occur at high elevation, and have human-mediated reductions in plant density due to harvesting. We found that two rare monocarpic Tibetan plant species, known as the Himalayan Snow Lotus, both require pollinators and that seed production in Saussurea medusa (Asteraceae) but not S. laniceps is limited by pollen receipt. We created deterministic and stochastic stage-structured matrix models for S. medusa, and found that pollen supplementation significantly increases population growth rate. However, even when pollen is not limiting, S. medusa is likely at risk for extinction in the next 50 years. Our results for this monocarpic plant differ from other population studies on pollen limitation in polycarpic plant species since the magnitude of pollen limitation for seed production was relatively low, and yet the sensitivity of population growth to changes in seed production was relatively high.     

Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L. (grapevine) suspension culture

Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins. AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V. vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation. An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating. The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx. 28–29%, with no apparent preference for anthocyanin species. This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2. The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Revealing differentially expressed proteins in two morphological forms of <Emphasis Type="Italic">Spirulina platensis</Emphasis> by proteomic analysis

Spirulina is distinguished from other cyanobacteria by its spiral morphology; however, this cyanobacterium has frequently been observed with a linear morphology in laboratory and industrial conditions. In our laboratory conditions, the simultaneously presence of the linear and spiral forms has also been observed. In the present study, the two forms of S. platensis C1 were separated and grown as axenic cultures in order to study the proteins that were differentially expressed in the soluble and insoluble protein fractions of the spiral and the linear forms. Two dimensional-differential gel electrophoresis (2D-DIGE) was performed to separate differentially expressed proteins that were subsequently identified by mass spectrometry. The differentially expressed proteins suggested two points. First, the morphological change is possibly induced by various environmental stresses such as oxygen level, carbon dioxide level, nutrient availability, and light. Second, the change of cell-shape might be a result of the change in a cell shape determination mechanism. Thus, this study is the first to show evidence at the protein level that may explain this morphological transformation in Spirulina.