Bioreactor shoot cultures of <Emphasis Type="Italic">Rhododendron tomentosum</Emphasis> (<Emphasis Type="Italic">Ledum palustre</Emphasis>) for a large-scale production of bioactive volatile compounds

Rhododendron tomentosum Harmaja (Ledum palustre), a peat bog plant from Ericaceae family, has been used in traditional medicine as the anti-arthritis agent. Although modern researches confirm its anti-inflammatory properties, it remains threatened by habitat degradation and possibilities to collect this endangered species from its natural environment for further biological activity studies are limited. Therefore, R. tomentosum liquid in vitro cultures were established as the alternative source of that valuable plant material. Schenk–Hildebrandt medium with 24.60 μM 2-isopentenyladenine and 592.02 μM adenine provides intensive growth and proper morphology of the obtained microshoots. The R. tomentosum biomass was scaled up using the various bioreactors (immersion, temporary immersion and spraying systems) for better growth and improved volatile oil production. The largest biomass accumulation (fresh weight?=?250 g l^?1, growth index?=?280, dry weight?=?20 g l^?1) and essential oil content (0.5% v/m) were achieved with application of commercially available RITA^® bioreactor. GC/MS analysis revealed the high content of p-cymene (6.9%), alloaromadendrene (5.5%), shyobunone (8.2%) and ledene oxide (II) (13.0%) in the volatile fraction obtained from RITA^® system. The biomass growth parameters and production profile in terms of essential oil and selected terpenoid compounds were determined during the 2 month period. The influence of culture conditions and bioreactor construction on the growth and volatile oil production in R. tomentosum biomasses was discussed.     

Effect of soil application of humic acid on nutrients uptake,essential oil and chemical compositions of garden thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) under greenhouse conditions

Humic acid is natural biological organic, which has a high effect on plant growth and quality. However, the mechanisms of the promoting effect of humic acid on the volatile composition were rarely reported. In this study, the effects of soil application of humic acid on the chemical composition and nutrients uptake of Thymus vulgaris were investigated. Treatments comprised 0, 50, 75 and 100 g m^?2. Essential oil was extracted by hydrodistillation and analyzed using GC–MS and GC–FID. Essential oil content was enhanced by increase of the humic acid level and its content ranged from 0.8% (control) to 2.0% (75 g m^?2). Thirty-two volatile compounds were identified and these compounds were considerably affected by humic acid. The highest percentage of thymol (74.15%), carvacrol (6.20%), p-cymene (4.24%), borneol (3.42%), trans-caryophyllene (1.70%) and cis-sabinene hydrate (1.35%) as major compounds were observed in T. vulgaris under 100 g m^?2 humic acid. There was a linear relationship (R² = 97%) between humic acid levels and thymol as a major compound. The oils were dominated by oxygenated monoterpenes followed by monoterpene hydrocarbons and sesquiterpene hydrocarbons. Based on the path coefficient analysis, the highest direct effects on essential oil content were observed in monoterpene esters (3.465) and oxygenated sesquiterpenes (3.146). The humic acid application also enhanced the uptake of N, P, K, Mg and Fe in garden thyme. The highest N (2.42%), P (0.75%), K (2.63%), Mg (0.23%) and Fe (1436.58 ppm) were observed in medium supplemented with 100 g m^?2 humic acid. In all, the utilization of humic acid could positively change nutrients uptake, essential oil content and its major constituents in T. vulgaris.     

Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Antitumor and antioxidant activity of the freshwater macroalga <Emphasis Type="Italic">Cladophora surera</Emphasis>

Macroalgae are currently being explored as novel and sustainable sources of bioactive compounds for both pharmaceutical and nutraceutical applications arising from their antioxidant, anticancer, and antimicrobial activity. In the present study, the antitumoral and antioxidant activities of crude methanolic extracts of the freshwater macroalga Cladophora surera Parodi & Cáceres, harvested from Napostá Creek (Argentina), were investigated in vitro. The antioxidant activity was assessed by DPPH method and polyphenol content using Folin-Ciocalteu phenol reagent. Antitumoral activity was evaluated on the human breast adenocarcinoma cell line MCF-7 by measuring proliferation, migration, and cell adhesion. The algal extract (AE) showed a total phenol content of 1.62?±?0.17 μg GAE mg^?1 dry alga and DPPH scavenging activity of 25.03?±?1.99% (10 mg)^?1 dry alga. The trypan blue assay after 48 h of treatment indicated that the AE significantly inhibits proliferation in a dose-dependent manner (1–100 μg mL^?1), being more effective the highest dose employed, with a concomitant increment in dead cells. However, the colorimetric MTS assay only showed a significant decrease in cell viability at 100 μg mL^?1 AE. Using the wound healing assay, we demonstrated that AE inhibits cell migration. Through a cell adhesion assay, we found that AE affects considerably the cell adhesion capacity at all doses probed. Analysis of cell spreading indicated that cell morphology was also affected by AE treatment. These results indicate that C. surera could be a source of valuable bioactive compounds usable as antitumoral preventive therapy for their effects on the regulation of processes involved in metastasis in cells derived from human mammary cancer.     

Morpho-physiological and phytochemical traits of (<Emphasis Type="Italic">Thymus daenensis</Emphasis> Celak.) in response to deficit irrigation and chitosan application

Thymus daenensis Celak. is an aromatic herb used as a popular medicine and its natural products in the form of extracts and essential oil have significant economic values in Iran. We hypothesized that spraying plants grown under deficit irrigation system with chitosan can be considered as an applicable method to enhance essential oil and antioxidant activity in thyme. Response of thyme to three irrigation regimes including well-watered, moderate stress, and severe stress along with three levels of chitosan application rates 0, 200, and 400 μL L^?1 was evaluated in a 2-year study in 2014 and 2015. Drought stress condition significantly shortened phenologic stages, more specifically in the first (establishment) year. All growth parameters were reduced dramatically as drought stress intensified. Imposing even moderate stress reduced leaf area as much as 59 and 44% in the first year and the second year, respectively. Biomass yield of plants grown under severe drought stress decreased substantially, whereas essential oil content and the share of thymol in thyme oil which possesses the greatest degree of biological activity improved. Maximum oil yield (1.50 g plant^?1) was obtained from plants under mild drought stress when sprayed with 400 μL L^?1 chitosan in the second year when plants were well-established. Foliar applications of chitosan reduced the adverse effect of water deficit on oil yield and improved thymol content of the essential oil. Chitosan also increased secondary metabolites including α-terpinene, p-cymene, γ-terpinene, thymol, carvacrol and β-caryophyllene. Leaf flavonoid reduced under deficit irrigation while more phenol was found in plants grown under deficit irrigation. The essential oil of thyme exhibited antioxidant property when the plants were sprayed with 400 μL L^?1 chitosan. The results of this study indicated that thyme can be grown successfully under moderate stress and that application of chitosan elicitor can to some degree compensate the negative impact of deficit irrigation on its biomass and essential oil yield.     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Terpenoids dominate the bouquet of volatile organic compounds produced by <Emphasis Type="Italic">Passiflora edulis</Emphasis> in response to herbivory by <Emphasis Type="Italic">Heliconius erato phyllis</Emphasis> (Lepidoptera: Nymphalidae)

In response to injury, plants produce volatile organic compounds (VOCs) that usually differ depending on the type of damage they have suffered (e.g., mechanical damage, herbivory, and oviposition). The objectives of this study were to identify and compare the bouquet of volatiles emitted by passion vine plants (Passiflora edulis) after injury caused by mechanical damage (MD), herbivory (HB), and oviposition (OV) by the lepidopteran, Heliconius erato phyllis. Following injury, extracts of plant emissions were collected from each treatment every 24 h for three days and were analyzed by GC and GC/MS. Results show that plants emitted 12 volatiles before and after damage, namely terpenoids, ketones, and aldehydes. Although no significant differences were detected between the three treatments individually, if the entire bouquet of volatiles is analyzed, samples collected at 24 h were different from samples collected at 48 and 72 h. However, terpenoid emission increased significantly in HB plants after 24 h. HB plants emitted approximately 6300, 50, 46, 11, 6, and 3.6 times more (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (E)-β-ocimene, (Z)-β-farnesene, (E)-β-caryophyllene, and farnesane, respectively, compared to control plants. OV plants displayed a peak of emission of (E)-β-ocimene after 72 h, which distinguished them from HB plants. MD plants showed a general increase of VOCs versus undamaged control plants. Furthermore, it has been suggested that (E)-β-ocimene may be sequestered by larvae of H. erato phyllis as a component of the odoriferous bouquet of the abdominal scent glands present in adult males, which play a role in sexual communication.     

Production system influences volatile biomarkers in tomato

Introduction

In recent years, growers have used various production types, including high-tunnel systems, to increase the yield of tomatoes (Lycopersicon esculentum). However, the effect of high-tunnel cultivation, in comparison to conventional open-field production, on aroma and flavor volatiles is not fully understood.

Objectives

To optimize the extraction and quantification conditions for the analysis of tomato volatiles using headspace solid phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS), and study the effect of production systems on volatile profiles using metabolomics approach.

Methods

The HS-SPME conditions were optimized for extraction and GC–MS was used to quantify the volatiles from four tomato varieties grown in open-field and high-tunnel systems. Univariate and multivariate analyses were performed to identify the influence of production system on tomato volatiles.

Results and conclusions

The extraction of 2 g tomato samples at 60 °C for 45 min using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber gave the maximum amounts of volatiles. This optimized method was used to identify and quantify 41 volatiles from four tomato varieties. The levels of β-damascenone were higher in the high-tunnel tomatoes and geranylacetone was higher in open-field tomatoes. These two volatile compounds could be considered as biomarkers for tomatoes grown in high-tunnel and open-field production systems. This study is the first report comparing volatiles in tomatoes grown in high-tunnel and open-field conditions, and our results confirmed that there is a critical need to adopt biomarker-specific production systems to improve the nutritional and organoleptic properties of tomatoes.

     

Microbial community and physicochemical dynamics during the production of ‘Chicha’, a traditional beverage of Indigenous people of Brazil

The microbial community of artisanal corn fermentation called Chicha were isolated, purified and then identified using protein profile by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) and confirmed by partial ribosomal gene sequencing. Samples from Chicha beverage were chemically characterized by gas and liquid chromatography (HPLC and GC-MS). Aerobic mesophilic bacteria (AMB) (35.8% of total of isolated microorganisms), lactic acid bacteria (LAB) (21.6%) and yeast (42.6%) were identified. Species of the genera Klebsiella, Bacillus, Staphylococcus, Micrococcus, Enterobacter, and Weissella were identified. Rhodotorula mucilaginosa, Lodderomyces elongisporus, Candida metapsilosis, and C. bohicensis were the yeasts found. The LAB isolates detected were responsible for the high concentrations of lactic acid found during the fermentation process (1.2 g L^??1), which is directly related to the decrease in pH values (from 6.95 to 3.70). Maltose was the main carbohydrate detected during corn fermentation (7.02 g L^??1 with 36 h of fermentation). Ethanol was found in low concentrations (average 0.181 g L^??1), making it possible to characterize the beverage as non-alcoholic. Twelve volatile compounds were identified by gas chromatography; belonging to the groups acids, alcohols aldehydes, acetate and others. MALDI-TOF was successfully used for identification of microbiota. Weissella confusa and W. cibaria were detected in the final product (after 36 h of fermentation), W. confusa is often classified as probiotic and deserve further application studies.     

Cultivation of <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> in anaerobically treated brewery wastewater for cost-effective biodiesel production

The use of wastewater has been investigated to overcome the economic challenge involved with a production of microalgae-based biodiesel. In this study, to achieve economical biodiesel production along with effective wastewater treatment at the same time, anaerobically treated brewery wastewater (ABWW) was utilized as a low-cost nutrient source, in the cultivation of Chlorella protothecoides. About 96 and 90 % of total nitrogen and phosphorus in ABWW were removed, respectively, while C. protothecoides was accumulating 1.88 g L^?1 of biomass. The C. protothecoides grown in ABWW showed increases in cell size and cell aggregation, resulting in a near 80 % enhanced harvesting efficiency within 20 min, as compared with only 4 % in BG-11. In addition, the total fatty acid content of the C. protothecoides grown in ABWW increased by 1.84-fold (35.94 ± 1.54 % of its dry cell weight), relative to that of BG-11.     

Isolation and characterization of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> LY214, a camptothecin-producing endophytic bacterium from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptothecin (CPT) is mainly produced and extracted from Camptotheca acuminata and Nothapodytes foetida for pharmaceutical use, i.e., the starting material for chemical conversion to the clinical CPT-type drugs. As the third largest plant anticancer drug, the heavy demand on CPT from global market leads to many research efforts to identify new sources for CPT production. Herein we report the isolation and characterization of a CPT-producing endophytic bacterium Paenibacillus polymyxa LY214 from Camptotheca acuminata. A 10.7 μg l^?1 of CPT was presented in the fermentation broth of P. polymyxa LY214. Its CPT production decreased sharply when the strain of the 2nd generation of P. polymyxa LY214 was cultured and fermented. However, the CPT production remained relatively constant from 2.8 μg l^?1 of the 2nd generation to 0.8 μg l^?1 of the 8th generation of P. polymyxa LY214 under optimized fermentation conditions. A 15- to 30-fold increase of CPT yield was observed when the optimized fermentation conditions, together with the addition of putative biosynthetic precursors of CPT and adsorbent resin XAD16, were applied to ferment the strains of the 7th and 8th generation of P. polymyxa LY214. Bioinformatics analysis of the relative species of P. polymyxa LY214 indicates its potential to produce CPT, which will be helpful to decipher the mysteries of CPT biosynthesis.     

A novel amidase from <Emphasis Type="Italic">Brevibacterium epidermidis</Emphasis> ZJB-07021: gene cloning,refolding and application in butyrylhydroxamic acid synthesis

A novel amidase gene (bami) was cloned from Brevibacterium epidermidis ZJB-07021 by combination of degenerate PCR and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The deduced amino acid sequence showed low identity (≤55 %) with other reported amidases. The bami gene was overexpressed in Escherichia coli, and the resultant inclusion bodies were refolded and purified to homogeneity with a recovery of 22.6 %. Bami exhibited a broad substrate spectrum towards aliphatic, aromatic and heterocyclic amides, and showed the highest acyl transfer activity towards butyramide with specific activity of 1331.0 ± 24.0 U mg^?1. Kinetic analysis demonstrated that purified Bami exhibited high catalytic efficiency (414.9 mM^?1 s^?1) for acyl transfer of butyramide, with turnover number (K _cat) of 3569.0 s^?1. Key parameters including pH, substrate/co-substrate concentration, reaction temperature and catalyst loading were investigated and the Bami showed maximum acyl transfer activity at 50 °C, pH 7.5. Enzymatic catalysis of 200 mM butyramide with 15 μg mL^?1 purified Bami was completed in 15 min with a BHA yield of 88.1 % under optimized conditions. The results demonstrated the great potential of Bami for the production of a variety of hydroxamic acids.     

Process Engineering for High-Cell-Density Cultivation of Lipid Rich Microalgal Biomass of <Emphasis Type="Italic">Chlorella</Emphasis> sp. FC2 IITG

In the present study, process engineering strategy was applied to achieve lipid-rich biomass with high density of Chlorella sp. FC2 IITG under photoautotrophic condition. The strategy involved medium optimization, intermittent feeding of limiting nutrients, dynamic change in light intensity, and decoupling growth and lipid induction phases. Medium optimization was performed using combinations of artificial neural network or response surface methodology with genetic algorithm (ANN-GA and RSM-GA). Further, a fed-batch operation was employed to achieve high cell density with intermittent feeding of nitrate and phosphate along with stepwise increase in light intensity. Finally, mutually exclusive biomass and lipid production phases were decoupled into two-stage cultivation process: biomass generation in first stage under nutrient sufficient condition followed by lipid enrichment through nitrogen starvation. The key findings were as follows: (i) ANN-GA resulted in an increase in biomass titer of 157 % (0.95 g L^?1) in shake flask and 42.8 % (1.0 g L^?1) in bioreactor against unoptimized medium at light intensity of 20 μE m^?2 s^?1; (ii) further optimization of light intensity in bioreactor gave significantly improved biomass titer of 5.6 g L^?1 at light intensity of 250 μE m^?2 s^?1; (iii) high cell density of 13.5 g L^?1 with biomass productivity of 675 mg L^?1 day^?1 was achieved with dynamic increase in light intensity and intermittent feeding of limiting nutrients; (iv) finally, two-phase cultivation resulted in biomass titer of 17.7 g L^?1 and total lipid productivity of 313 mg L^?1 day^?1 which was highest among Chlorella sp. under photoautotrophic condition.     

Volatile essential oil chemical composition of basil (<Emphasis Type="Italic">Ocimum basilicum</Emphasis> L. ‘Green’) cultivated in a greenhouse and micropropagated on a culture medium containing copper sulfate

The objective of this study was to determine the effects of copper sulfate (CuSO₄) on the chemical composition of basil (Ocimum basilicum L. ‘Green’) using static headspace extraction. The basil was cultivated in vitro and ex vitro. The sowing was completed in trays, and the seedlings were transplanted to pots and grown in a protected environment for 180 d. For in vitro cultivation, the seeds were placed on Murashige and Skoog (MS) medium enriched with growth regulators, sucrose, agar, and CuSO₄ (at 0 μM [control], 25 μM, or 75 μM). Volatile organic compounds emitted from the excised leaves were collected by the static headspace technique, and identified by gas chromatography coupled to mass spectrometry (GC/MS). Twenty-six compounds were identified in the leaves harvested from the plants cultivated in vitro, while 11 compounds were identified in the leaves sampled from the ex vitro plants. Oxygenated monoterpenes were the main compounds found in plants cultivated ex vitro. Phenylpropanoids predominated in the control and the 25 μM CuSO₄ treatments. The main compounds found were methyl eugenol (52.03%) and eugenol (20.66%). For the 75 μM CuSO₄ treatment, the major compounds detected were linalool (28.14%) and 1.8-cineole (15.7%). Volatile secondary metabolites of basil cultivated in vitro with CuSO₄ were easily isolated and rapidly obtained. The results of this study demonstrate the feasibility and potential of using copper treatments to reduce the impact of seasonality on essential oil production.     

An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Improvement of biomass and fatty acid productivity in ocean cultivation of <Emphasis Type="Italic">Tetraselmis</Emphasis> sp. using hypersaline medium

In this study, hypersaline media were used for ocean cultivation of the marine microalga Tetraselmis sp. KCTC12432BP for enhanced biomass and fatty acid (FA) productivity. Hypersaline media (55, 80, and 105 PSU) were prepared without sterilization by addition of NaCl to seawater obtained from Incheon, Korea. The highest biomass productivity was obtained at 55 PSU (0.16 g L^?1 day^?1) followed by 80 PSU (0.15 g L^?1 day^?1). Although the specific growth rate of Tetraselmis decreased at salinities higher than 55 PSU, prevention of contamination led to higher biomass productivity at 80 PSU than at 30 PSU (0.03 g L^?1 day^?1). FA content of algal biomass increased as salinity increased to 80 PSU, above which it declined, and FA productivity was highest at 80 PSU. Ocean cultivation of Tetraselmis was performed using 50-L tubular module photobioreactors and 2.5-kL square basic ponds, closed- and open-type ocean culture systems, respectively. Culturing microalgae in hypersaline medium (80 PSU) improved biomass productivities by 89 and 152% in closed and open cultures, respectively, compared with cultures with regular salinity. FA productivity was greatly improved by 369% in the closed cultures. The efficacy of salinity shift and N-deficiency to enhance FA productivity was also investigated. Lowering salinity to 30 PSU with N-starvation following cultivation at 80 PSU improved FA productivity by 19% in comparison with single-stage culture without N-deficiency at 30 PSU. The results show that salinity manipulation could be an effective strategy to improve biomass and FA productivity in ocean cultivation of Tetraselmis sp.     

Isolation and characterization of trichalasin-producing endophytic fungus from <Emphasis Type="BoldItalic">Taxus baccata</Emphasis>

An endophytic fungus (strain T1) isolated from Taxus baccata was studied for the production of metabolites with anticancer and antioxidant activities. This fungus was identified as Diaporthe sp. based on rDNA-internal transcribed spacer (ITS) sequence analysis. The crude extract showed cytotoxic activity against MCF-7 and HeLa cancer cell lines, with IC₅₀ (concentration inhibiting 50% of growth rate) values of 1058?±?44 and 1257?±?80 μg ml^?1, respectively. The scavenging activity of fungal extract increased significantly with increasing concentration [IC₅₀ (concentration required to scavenge 50% of free radicals) 482?±?9 μg ml^?1]. Ultra-high-performance liquid chromatography-quadrupole-time of flight analysis revealed the presence of three trichalasins (trichalasin E, F and H) in the crude extract of T1 which are known to have antitumour and antioxidant activities. These results suggest that Diaporthe sp. has the potential to be used for therapeutic purposes because of its antiproliferative and antioxidant potential and also for the production of cytochalasins.