Secondary chemistry of cultured mycobionts: formation of a complete chemosyndrome by the lichen fungus of Lobaria spathulata

Abstract:The study aimed to optimize culture conditions and nutrient requirements for the production of secondary metabolites by the cultured mycobiont Lobaria spathulata. This species proved to be an excellent model system for such studies, as the complete chemosyndrome found in the natural lichen thallus was repeatedly formed in the cultured mycobiont with differentiated, aerial mycelia. Nutrient media containing the disaccharide, sucrose, were found to favour both rapid growth and the production of typical lichen substances. Higher proportions of the secondary compounds were detected in the developing mycobiont than in mature lichen thalli.     

Culture studies on the mycobiont isolated from Parmotrema reticulatum (Taylor) Choisy: metabolite production under different conditions

A strain of the lichen mycobiont isolated from a thallus of Parmotrema reticulatum was cultured axenically on different media. The morphology, anatomy, growth of the colonies, and metabolite production were studied. The isolated fungal colonies developed well and showed a remarkable morphogenetic capacity on most of the assayed solid media, e.g., malt extract 2%-yeast extract 0.2% (MEYE), malt extract 1%-yeast extract 0.4%-sucrose 10% (MY10), and the original Lilly & Barnett medium (LB). The identity of the isolated fungus was confirmed by its ITS rDNA-sequence. Atranorin, the major cortical lichen depside, was produced when the colonies were grown over 5 and 10 months on solid LB medium, combined with a dessication treatment. Atranorin was identified by matching of UV spectra obtained from HPLC running and a reference substance in a spectrum library. Colonies grown on MEYE and MY10 with a dessication treatment did not produce any lichen secondary metabolite. Mycobionts grown for 5 months on solid MEYE without a dessication treatment produced triacylglycerides as the major metabolites, and the fatty acids were characterized as their methyl esters. Analysis by TLC and HPLC-DAD of extracts of colonies grown on LB and MY10 without dessication revealed that the typical secondary compounds of the natural lichen were not produced. The major metabolites of the natural lichen thallus were identified by chromatographic and spectroscopic methods.     

Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of <Emphasis Type="Italic">Artemisia absinthium</Emphasis> L.

Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Algal and Fungal Diversity in Antarctic Lichens

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7–100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen‐associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen‐associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen‐associated fungi was not evident.     

Establishment of culturing conditions and assessment of antioxidant activity and somaclonal variation in the adventitious root suspension cultures of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> Nakai

Oplopanax elatus Nakai, a plant traditionally used in folk medicine, is currently in population decline due to uncontrolled harvesting. In the present study, we investigated the factors affecting O. elatus adventitious root production, including hormones (alone or in combination), explant type, basal salt type and strength, sucrose concentration, pH, and temperature. Results revealed that adventitious root formation was optimal with root explants grown on 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^?1 Indole-3-butyric acid (IBA) (pH 5.8) at 25 °C. Chlorogenic acid concentration was highest in roots propagated in 1/2 MS medium containing 0.5 mg L^?1 IBA; vanillin, another phenolic compound, was also detected in cultures. Liquid media containing 3% sucrose exhibited the highest radical scavenging activity and total phenolic compound contents. X-ray diffraction revealed significant differences in the elemental intensity between adventitious root and field-grown plantlet extracts. Analysis of simple sequence repeats confirmed that adventitious roots regenerated in vitro were genetically similar to their mother plant. Thus, we identified the optimal conditions for proliferation of O. elatus adventitious roots in liquid culture, from which, secondary metabolites, particularly bioactive compounds associated with the medicinal use of this plant, can be mass produced without further population deterioration.     

Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives

In an attempt to scale-up of adventitious root cultures of Echinacea angustifolia for the production of biomass and caffeic acid derivatives, i.e. echinacoside, chlorogenic acid, cichoric acid, caftaric acid, and cynarin, the effects of Murashige and Skoog (MS) medium dilutions, and initial sucrose concentrations were investigated in a 5-L airlift bioreactor. In addition, the kinetics of adventitious root growth and accumulation of secondary metabolites were also studied. The greatest root dry weight (6.50 g L^?l) and accumulation of total phenolics [22.06 mg g^?1 DW (dry weight)], total flavonoids (5.77 mg g^?1 DW) and total caffeic acid derivatives (10.63 mg g^?1 DW) were obtained at quarter-strength MS medium. Of the various gradients of sucrose tested, 5 % sucrose supplementation was regarded as an optimal concentration for enhancing productivity of biomass and bioactive compounds. Neither higher salt strength (3/4–2 MS) nor sucrose concentrations (7 and 9 %) showed promotive effect on root growth and metabolite production. The kinetic studies revealed that 4 weeks of culture period is the optimal time to achieve highest productivity of metabolites. Based on these results, a large-scale (20 L) and a pilot-scale (500 L) adventitious root culture system was established. In the pilot-scale bioreactor, adventitious roots were elicitor-treated with 100 μM methyl jasmonate (MJ) on day 28. After 1 week of elicitation, 1.75 kg dry root biomass was harvested containing 60.41 mg g^?1 DW of total phenolics, 16.45 mg g^?1 DW of total flavonoids, and 33.44 mg g^?1 DW of total caffeic acid derivatives. Among the caffeic acid derivatives, the accumulation of echinacoside (the major bioactive compound) in MJ-treated adventitious roots grown in the 500-L bioreactor was the highest (12.3 mg g^?1 DW), which is approximately threefold more than the non-MJ-treated roots cultured in 5- and 20-L bioreactors.     

Accumulation of rosmarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L.

Eryngium planum L. cell and organ cultures were maintained on Murashige and Skoog media (MS), supplemented with exogenous hormones of different types and various concentrations for high biomass growth. The callus and cell suspension cultures were treated with increased sucrose concentration and/or elicited by methyl jasmonate for the enhancement of selected phenolic acids accumulation. Three phenolic acids, rosmarinic acid (RA), chlorogenic acid (CGA) and caffeic acid (CA), were detected by HPLC-DAD in those cultures. The sum of their content in the dry material was found to be higher in the shoot culture (3.95 mg g^?1), root culture (7.05 mg g^?1), callus (6.20 mg g^?1) and cell suspension (2.04 mg g^?1) than in the leaves (1.87 mg g^?1) and roots (0.76 mg g^?1) of intact plants. The major compound of in vitro cultures was always rosmarinic acid. The content of RA could be increased approximately threefold (16.24 mg g^?1) in the callus culture and approximately twofold (3.91 mg g^?1) in the cell suspension culture by elicitation with 100 μM methyl jasmonate (MeJA). The higher concentration of sucrose (S) in the medium (5, 6 %) led to over a twofold increase of CGA content in the callus culture (2.54 mg g^?1). The three mentioned phenolic acids have been found in E. planum undifferentiated and differentiated in vitro cultures for the first time.     

Impacts of methyl jasmonate and phenyl acetic acid on biomass accumulation and antioxidant potential in adventitious roots of <Emphasis Type="Italic">Ajuga bracteosa</Emphasis> Wall ex Benth., a high valued endangered medicinal plant

Ajuga bracteosa is a medicinally important plant globally used in the folk medicine against many serious ailments. In the present study, effects of two significant elicitors, methyl jasmonate (Me-J) and phenyl acetic acid (PAA) were studied on growth parameters, secondary metabolites production, and antioxidant potential in adventitious root suspension cultures of A. bracteosa. The results showed a substantial increase in biomass accumulation, exhibiting longer log phases of cultures growth in response to elicitor treatments, in comparison to control. Maximum dry biomass formation (8.88 DW g/L) was recorded on 32nd day in log phase of culture when  0.6 mg/L Me-J was applied; however, PAA at 1.2 mg/L produced maximum biomass (8.24 DW g/L) on day 40 of culture.  Furthermore, we observed the elicitors-induced enhancement in phenolic content (total phenolic content), flavonoid content (total flavonoid content) and antioxidant activity (free radical scavenging activity) in root suspension cultures of A. bracteosa. Application of 0.6 mg/L and 1.2 mg/L of Me-J, root cultures accumulated higher TPC levels (3.6 mg GAE/g DW) and (3.7 mg GAE/g DW) in the log phase and stationary phase, respectively, while 2.5 mg/L Me-J produced lower levels (1.4 mg GAE/g DW) in stationary phase of growth stages. Moreover, TFC and FRSA values were found in correspondence to TPC values in the respective growth phases at the similar elicitor treatment. Thus, a feasible protocol for establishment of adventitious roots in A. bracteosa was developed and enhancement in biomass and metabolite content in adventitious root was promoted through elicitation.     

Secondary metabolites from isolated lichen mycobionts cultured under different osmotic conditions

Growth of the mycobiont of Ramalina siliquosa and the secondary metabolites subsequently produced under various osmotic culture conditions were examined. The secondary metabolite content and the growth rate changed greatly when different quantities of sucrose were added to the culture medium. Salazinic acid was found only in mycobionts cultured on a medium with 10 or 20% sucrose, the mycobiont growth rate being higher than on conventional medium. Similarly, in Lobaria discolor, gyrophoric acid was found only in mycobionts cultured on a medium with 10% sucrose.     

Accumulation of anthocyanin in callus cultures of red-pod okra [<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m^??2 s^??1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47?±?0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m^??2 s^??1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.     

Cell culture establishment and regulation of two phenylethanoid glycosides accumulation in cell suspension culture of desert plant <Emphasis Type="Italic">Cistanche tubulosa</Emphasis>

Cistanche tubulosa is one of the most valuable desert medicinal plants, whose cell culture investigations have been rarely reported before. Phenylethanoid glycosides (PhGs) are its major components with a wide range of pharmacological activities. In this article, callus culture and cell suspension of C. tubulosa were established. Fleshy stems were found to be the most suitable explants for callus induction, and the optimal medium for induction was B5 solid medium supplemented with 0.8 g/L casein hydrolysate, 20 g/L sucrose, 2 mg/L naphthaleneacetic acid (NAA), and 1 mg/L 6-benzyladenine (6-BA). Based on qualitative and quantitative determination of two PhGs (echinacoside and acteoside) contents, the effects of carbon source concentration, precursor feeding, and elicitor treatments on cell growth and two PhGs accumulation in cell suspension cultures were investigated. Thirty g/L was the optimal initial sucrose concentration to obtain the high yield of biomass (9.29 g dry weight, DW) per liter cell suspension culture, echinacoside (12.14%, based on DW cells) and acteoside (2.17%). Precursor feeding also had a positive effect on PhGs accumulation. Feeding of precursor tyrosine (1 g/L) to the cell cultures increased the levels of echinacoside to 18.83% and acteoside to 2.92%, which were approximate 1.5 times of the corresponding levels in the control group. Methyl jasmonate (MJ) was the ideal elicitor for PhGs accumulations in C. tubulosa, particularly for eliciting acteoside production. The maximum echinacoside and acteoside contents reached 21.18 and 5.24% after 12 h of treatment with 200 µM MJ, respectively, which were approximate twofold higher than those in wild plant.     

Efficient enhancement of gallic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Barringtonia racemosa</Emphasis> L. by elicitation

The bioactive compound, bacoside A, has immense importance for the treatment of memory disorders and Alzheimer’s disease. Due to the growing commercial interest in the herb, Bacopa monnieri, it has been listed as highly endangered species. The present study was aimed at enhancing the production of bacoside A using an alternative technology of plant cell suspension culture. Initial experiments of docking simulations using bacoside A showed good inhibition of acetyl cholinesterase (binding energy value of ??20 kcal/mol), when comparison was made with other phytocompounds and the synthetic drug for Alzheimer’s disease. In vitro experiments established that B. monnieri cell suspension culture can be developed in Murashige and Skoog medium containing containing 0.1 mg/L benzylaminopurine and 0.5 mg/L naphthalene acetic acid. Plackett–Burman studies predicted that the most effective factors for maximum biomass production were inoculum size (t-value of 4.87), sucrose concentration (t-value of 0.25) and KH₂PO₄ concentration (t-value of 0.007). The nitrate to ammonium ratio (t-value of ? 0.42) did not have significant effect on the cell suspension biomass. The optimum concentration of the crucial variables obtained from a central composite design were—inoculum size of 2 g/L, sucrose concentration of 30 g/L and KH₂PO₄ concentration of 1.24 mM in one-sixth strength MS medium. The best model for optimum production of biomass and bacoside A was experimentally verified and the correlation between the predicted and actual values was found to be 99% for biomass and 94% for bacoside A production. The experimental results have been discussed in the present work.     

Adventitious root culture of <Emphasis Type="Italic">Polygonum multiflorum</Emphasis> for phenolic compounds and its pilot-scale production in 500 L-tank

Polygonum multiflorum Thunb. is an important medicinal plant that synthesizes an array of phenolic compounds. Its roots are used in a variety of pharmacological and cosmetic formulations, notably as hair dye. In the present study, the inoculum density (3–15 g/L) and culture period (1–7 weeks) were optimized in a 3 L bioreactor. High root biomass (14.18 g/L dry weight (DW)) was recorded with an inoculum of 7 g/L (p?≤?0.05), which is consistent with the results for 5 and 10 g/L. However, significantly higher yield of bioactive compounds (53.87 mg/g DW total phenolics and 27.96 mg/g DW total flavonoids) with high free radical scavenging activity was obtained in root samples from 5 g/L inoculum density. A 4 week culture period was sufficient for optimum root growth and metabolite production. The optimized conditions were used for large-scale (5 and 20 L) and pilot-scale (500 L) studies. Considering that the continuous aeration of root cultures may lead to oxidative stress, antioxidant enzyme activity and lipid peroxidation also were studied. The results revealed high catalase (CAT) and guaiacol peroxidase (G-POD) activities, and low malondialdehyde (MDA) production, with increasing culture scale (20 and 500 L), which may indicate low-level oxidative damage to the cultures. An optimal yield of 4.01 kg dry root biomass with 287.12 mg/L of total phenolic productivity was achieved in a 500 L pilot-scale bioreactor. This work can pave the way for commercial production of biomass and secondary metabolites at the industrial level, and meet the rising demand for natural ingredients, especially in the pharmaceutical and cosmetic industries.     

Alterations in secondary metabolism of aposymbiotically grown mycobionts of Xanthoria elegans and cultured resynthesis stages

HPLC analyses of Xanthoria elegans cultivated on different media and either aposymbiontically or with its photobiont revealed that the carbon source and the presence of the algal partner have an impact on the secondary metabolism of the mycobiont. The aposymbiotically (without photobiont) grown mycobiont contained up to 70% more of the main compounds in its thallus than in resynthesis stage. Although this is speculative, the induction of the polyketide pathway may be a feedback mechanism to the absence of the photobiont. All cultures produce a variety of substances which were not detectable in the voucher specimen. Besides physcion (the major substance), we were able to identify emodin as well as physcion-bisanthrone, teloschistin monoacetate and derivatives. A strong inducible effect on the production of physcion, physcion-bisanthrone and on their precursors and derivatives was found for mannitol. By contrast, supplementation of ribitol had negligible effects, if any, on polyketide quantities although it is the main carbon source for the mycobiont in free-living lichens with Trebouxia photobiont.     

Production of potential anti-inflammatory compounds in cell suspension cultures of <Emphasis Type="Italic">Sphaeralcea angustifolia</Emphasis> (Cav.) G. Don

Sphaeralcea angustifolia is used in Mexican traditional medicine to treat inflammatory processes. SCopoletin (SC), TOmentin (TO), and sphaeralcic acid (SA) were reported as the main anti-inflammatory compounds in this species. The aim of this study was to establish in vitro conditions for the development of calli and cell suspension cultures that are the producers of these active compounds. Callus cultures of plant leaf explants were set up using different auxin levels of α-naphthalene acetic acid (NAA) in combination with a constant concentration (0.1 mg L^?1) of Kinetin (Kn) in Murashige and Skoog (MS) medium. Optimal combinations for callus induction were 1.0 and 2.0 mg L^?1 of NAA. SC, TO, and SA were not detected in callus tissues. Employing a 4 % inoculum in fresh biomass, cell suspension was established from friable callus with 1.0 mg L^?1 of NAA in combination with 0.1 mg L^?1 of Kn in MS liquid medium (27.4 mM nitrate). The cellular suspension synthesized SC and SA, SC was excreted into the culture medium, while SA was excreted into the culture medium and accumulated in biomass. To improve SC and SA production, total nitrate content was reduced in MS medium. On diminishing nitrate content to 2.74 mM, cellular suspension growth was not modified. SC concentration (0.04 %) was 60-fold higher than that detected in the wild plant (0.00067 %), TO was produced (0.096 %), and SA content (0.0036 %) was not improved. SA production in MS medium with 0.274 mM nitrate (0.004 %) was enriched 12-fold (0.0003 %) in relation to that of the wild plant. The anti-inflammatory effects at 5 h of intraperitoneal (i.p.) administration (100 mg per kg BW) of dichloromethane extracts from the medium (42 ± 3 %) and biomass (39 ± 9.3 %) of S. angustifolia cell suspensions cultivated in MS with 2.74 mM nitrate were similar. The effect of the biomass dichloromethane extract was dose dependent with a median Effective Dose (ED₅₀) of 137.63 mg per kg BW.     

Fungal melanins as a sun screen for symbiotic green algae in the lichen<Emphasis Type="Italic"> Lobaria pulmonaria</Emphasis>

The mycobiont of the high-light-susceptible forest lichen Lobaria pulmonaria was shown to deposit brown, melanic compounds in the outer layer of the upper cortex, depending on the long-term level of solar radiation in its natural habitat. Furthermore, pale thalli from a shady habitat produced melanic compounds after transplantation to a sunny habitat. This browning of the cortex appeared to be a physiologically active process, taking place only during periods with frequent hydration. Melanin production was slow. After transplantation, more than 1 year was needed for a shade-adapted thallus to reduce the cortical transmittance (230-1000 nm) to a similar level to that of naturally sun-exposed specimens. Melanic compounds acted as a sun screen, especially reducing UVB and UVA wavelengths, but also visible wavelengths, at the photobiont level. In the near infrared range, there was only a small difference in transmittance between shade- and sun-adapted cortices. A negative correlation was found between the natural light level and the cortical transmittance of wavelengths below 700 nm. However, previous studies have shown that even photobionts of melanic L. pulmonaria thalli are relatively susceptible to high-light exposure. Since melanins also increase the absorbance of solar energy for the whole thallus, it appears that what is gained in terms of UV- and light protection in melanic L. pulmonaria specimens may be offset by increased exposure to excess temperatures for this highly heat-susceptible lichen.