Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

Phosphorus Enhances Photosynthetic Storage Starch Production in a Green Microalga (Chlorophyta) Tetraselmis subcordiformis in Nitrogen Starvation Conditions.

Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway.

The characteristics of TAG and EPA accumulation in Nannochloropsis oceanica IMET1 under different nitrogen supply regimes.

The strategy of nitrogen limitation has been widely applied to enhance lipid production in microalgae. The changes of cellular composition, and the characteristics of triacylglycerol (TAG) and eicosapentaenoic acid (EPA) accumulation in Nannochloropsis oceanica IMET1 were investigated. The results revealed that after nitrogen limitation TAG rather than carbohydrate was the dominant carbon sink in N. oceanica IMET1. Different nitrogen supplementation strategies were applied in order to achieve high TAG and EPA productivity, respectively. Limited nitrogen was supplied to improve TAG production, and a maximum productivity of 29.44 mg L(-1) d(-1) was obtained, which was a 6.74-fold increase compared to nitrogen-depleted cultivation. The highest EPA productivity of 7.66 mg L(-1) d(-1) was achieved under nitrogen-replete cultivation, which is different from the condition for TAG maximum productivity because the EPA is in glycolipids and phospholipids mainly. The fatty acid composition analysis identified the source of acyl group in TAG accumulation.

Luteimonas dalianensis sp. nov., an obligate marine bacterium isolated from seawater.

A marine bacterial strain, designated OB44-3(T), was isolated from a crude oil-contaminated seawater sample collected near Dalian Bay, China. Cells of strain OB44-3(T) were Gramnegative, aerobic, rod-shaped, and oxidase- and catalasepositive. The major fatty acids were branched-chain saturated iso-C15:0 (27.9%) and unsaturated iso-C17:1 ω9c (14.8%). The DNA G+C content was 64.6 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain OB44-3(T) was a member of the genus Luteimonas (95-96% 16S rRNA gene sequence similarity); its closest neighbors were the type strains of Luteimonas terricola (96% sequence similarity), Luteimonas mephitis (96%), and Luteimonas lutimaris (96%). On the basis of phenotypic, chemotaxonomic, and phylogenetic distinctiveness, strain OB44-3(T) was considered to represent a novel species of the genus Luteimonas. The name Luteimonas dalianensis sp. nov. is proposed, with strain OB44-3(T) (=CGMCC 1.12191(T) =JCM 18136(T)) as the type strain.

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions.

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0×106 cells mL⁻¹. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1% was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L⁻¹ day⁻¹, starch concentration of 1.6 g L⁻¹, and starch productivity of 0.30 g L⁻¹ day⁻¹ were all obtained in the culture with the ICD of 3.0×106 cells mL⁻¹. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH2PO4 was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.

[Quantitative analysis of hinokiol from cell suspension cultures of Cephalotaxus fortunei].

A high performance liquid chromatography (HPLC) method was developed for the separation of secondary metabolites and quantitative analysis of hinokiol from cell suspension cultures of Cephalotaxus fortunei. The samples were prepared by extraction using methanol followed by partitioning between ammonium hydroxide and chloroform. The HPLC separation was achieved on an Apollo C18 column (250 mm x 4.6 mm, 5 microm) with gradient elution using methanol and water at 1 mL/min and 30 degrees C. The detection was carried out at 290 nm. A good linear correlation between the hinokiol peak area and mass concentration was observed over the mass concentration range of 0.012 5 - 0.2 g/L. The proposed method was applied to the determination of hinokiol in the actual samples with recoveries of 87.2% - 94.7% and with the relative standard deviations of 0.9% - 4.2%. This method is reliable and reproducible and is suitable for the analysis of hinokiol in plant cell cultures.

Comparison of spiculogenesis in in vitro ADCP-primmorph and explants culture of marine sponge Hymeniacidon perleve with 3-TMOSPU supplementation.

This study aims to test the feasibility of introducing functional chemical groups into biogenic silica spicules by examining the effect of supplementing a silican coupler [3-(trimethoxysilyl)propyl]urea (3-TMOSPU) as silica source in the cultures of archaeocytes-dominant-cell-population (ADCP) primmorphs and explants of the marine sponge Hymeniacidon perleve. Analysis by Fourier Transform Infrared Spectroscopy (FT-IR) confirmed that the organic group in 3-TMOSPU was introduced into silica spicules. By comparing ADCP-primmorph cultures when supplemented with Na2SiO3, 3-TMOSPU supplementation showed no notable effect on the primmorphs development and cell locomotion behaviors. A decline in silicatein expression quantified by real-time RT-PCR was, however, observed during spiculogenesis. The decline was slower for the 3-TMOSPU group whereas significantly fewer spicules were formed. When sponge papillae explants were cultured, 3-TMOSPU supplementation had no negative effect on sponge growth but inhibited the growth biofouling of the diatom Nitzschia closterium. By monitoring the detectable Si concentration, it seemed that 3-TMOSPU was converted by the sponge and its conversion was related to spiculogenesis. Analysis of spicule dimensional changes indicated that the inhibition of spiculogenesis by 3-TMOSPU supplementation was less in ADCP-primmorphs culture due to lower 3-TMOSPU/detectable Si ratio in the media.