Effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium phalae)

Summary Elicitation of anthocyanin-producing cells of ohelo (Vaccinium pahalae) by both biotic (purified β-glucan and chitosan) and abiotic [sodium ferric ethylenediamine di-(o-hydroxyphenylacetate) FeEDDHA, and CuSO₄] elicitors resulted in significant enhancement of anthocyanin accumulation. Anthocyanin production increased up to 1.8 and 1.5-fold over the control in the presence of abiotic elicitors (90 μM FeEDDHA and 20 μM CuSO₄, respectively), and increased 1.9 and 1.6-fold in the presence of biotic elicitors (10 mg L⁻¹ β-glucan and 100 mg L⁻¹ chitosan). Maximum anthocyanin production with the two most effective elicitors was achieved when cultures were treated on Day 3 (β-glucan) or Day 0 (FeEDDHA) after the initiation of fresh cell cultures. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). The addition of 0.5 μM MJ alone provoked a 2–3-fold increase in anthocyanin production over that of the control; however, no additive effect on anthocyanin production was observed in any treatments which combined MJ and β-glucan or FeEDDHA. Conditioning of the cells with a preculture in either MJ, β-glucan, or FeEDDHA similarly did not enhance anthocyanin production. Inoculation of cultures elicited by MJ or β-glucan with ibuprofen, a reported inhibitor of jasmonate biosynthesis, dramatically stimulated, rather than inhibited, anthocyanin production, resulting in levels of accumulation beyond any of the tested elicitor combinations. Hypotheses for the observed influence of ibuprofen in this system are discussed.     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.     

The effect of growth retardants on anthocyanin production in carrot cell suspension cultures

The effect of growth retardants on anthocyanin production was studied in wild carrot (Daucus carota) cell suspension cultures. Paclobutrazol [(2RS,3RS) — 1 — (4-chlorophenyl) — 4,4 —dimethyl-2-(1,2,4-triazol-1-yl) pentan-3-ol], uniconazole [(E)-1-(4-chlorophenyl-4,4 —) dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol], tetcyclacis [5-(4-chloro-phenyl) -3,4,5,9,10-pentaaza-tetracyclo-5, 4, 10^2,6, O^8,11 — dodeca-3, 9-diene], ancymidol [-cyclopropyl — 4 — methoxy-(pyrimidine-5-yl)benzyl alcohol] and CCC (2-chloro-ethyltrimethylammonium chloride) increased anthocyanin accumulation. AMO-1618 [(2-isopropyl-5-methyl-4-trimethyl-ammonium-chloride)-phenyl-1-piperidinium carboxylate] did not increase anthocyanin accumulation in the first passage but did increase it during the second passage on medium for improved anthocyanin accumulation. Prohexadione (3,5-dioxo-4-propionylcyclohexane carboxylic acid) decreased anthocyanin accumulation by 10%–12.5%.The inhibitory effect of gibberellin on anthocyanin accumulation was reversed by paclobutrazol. Paclobutrazol together with 10^–6M GA₃ increased anthocyanin level from 33% of control in GA₃ treated cell suspension to 76%. These results are consistent growth retardants increasing anthocyanin accumulation in carrot cell suspension cultures by inhibiting gibberellin biosynthesis.     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

Effects of abiotic stress on biomass and anthocyanin production in cell cultures of Melastoma malabathricum

Plant cell cultures could be used as an important tool for biochemical production, ranging from natural coloring (pigments) to pharmaceutical products. Anthocyanins are becoming a very important alternative to synthetic dyes because of increased public concern over the safety of artificial food coloring agents. Several factors are responsible for the production of anthocyanin in cell cultures. In the present study, we investigate the effects of different environmental factors, such as light intensity, irradiance (continuous irradiance or continuous darkness), temperature and medium pH on cell biomass yield and anthocyanin production in cultures of Melastoma malabathricum. Moderate light intensity (301 - 600 lux) induced higher accumulation of anthocyanins in the cells. The cultures exposed to 10-d continuous darkness showed the lowest pigment content, while the cultures exposed to 10-d continuous irradiance showed the highest pigment content. The cell cultures incubated at a lower temperature range (20 ± 2 oC) grew better and had higher pigment content than those grown at 26 ± 2 oC and 29 ± 2 oC. Different medium pH did not affect the yield of cell biomass but anthocyanin accumulation was highest at pH 5.25 - 6.25.     

Genetically controlled anthocyanin synthesis in cell cultures of Matthiola incana

Callus cultures were derived from different parts of 8 anthocyanin producing and 2 white flowering lines of the crucifer Matthiola incana. The tissue cultures of the cyanic lines were shown to produce genotype specific anthocyanin patterns, whereas in the calli of the acyanic lines no anthocyanin synthesis occured.Abbreviations IAA indoleacetic acid - 2,4-D dichlorophenoxyacetic acid - MeOH methanol - Et₂O ether - ETOAc ethylacetate     

High lipids accumulation in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by applying single and multiple nutrients limitation in a simple chemically defined medium

Microbial oil produced by the oleaginous yeast Rhodosporidium toruloides ATCC 204091 (formerly referred to as Rhodotorula glutinis) has a similar fatty acid composition to the vegetable oils and represents a potential alternative for biodiesel production. Finding strategies to improve the oil production by this yeast is desirable, as it is one of this nutrient’s limitations during the accumulation phase, as well as one of the main factors influencing the process. Therefore, the effect of single or combined nutrient limitation on lipid accumulation by R. toruloides was investigated. Biomass production and lipid accumulation by R. toruloides was improved using experimental designs in a two-step batch culture on a chemically-defined culture medium with high initial glucose concentration. For the first culture step, a Box–Behnken design was applied to optimize the main medium components’ concentrations, while maintaining a high biomass production. A biomass concentration of 44.3 g/L was reached with a medium composed of (g/L): glucose, 100; KH₂PO₄, 4.6; NaNO₃, 13.4; MgSO₄ ^.7H₂O, 0.2; and CaCl₂ ^.2H₂O, 0.11. For the second culture step, the biomass was transferred to lipid accumulation media. A 2³ factorial experimental design was conducted to investigate the effect of N, P and S limitations (individually or jointly) on lipid production from glucose (100 g/L). Lipid accumulation on dry cell mass was 77.04, 65.42, 70.13 and 69.84% for N, P, S and simultaneous nutrients’ limitations, respectively.     

Oxytocin influences the production of glycyrrhizin from cell cultures of Abrus precatorius Linn.

Oxytocin, a peptide animal hormone, was used as a growth regulator to test its effect on biomass accumulation and production of secondary plant constituent glycyrrhizin in the cell cultures of Abrus precatorius. Glycyrrhizin is an important phytoconstituent of liquorice which is widely used in the pharmaceutical and food industries. Cell suspension cultures of A. precatorius were developed from leaf explant of in vitro germinated plant in Murashige and Skoog medium supplemented with 30 g/l sucrose, 1 mg/l naphthalene acetic acid and 1 mg/l kinetin. The influence of oxytocin on biomass accumulation as well as on the production of glycyrrhizin was observed in the cell cultures of A. precatorius. Treatment of A. precatorius cell cultures with 100 μg/l oxytocin, improved glycyrrhizin production up to 34.27 mg/l on the dry cell weight basis third day after oxytocin treatment, which is over four times that of the control cultures, simultaneously nearly two fold increase in the biomass 2 days after the oxytocin treatment was recorded over the control cultures.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Localization changes of endogenous hydrogen peroxide during cell division cycle of Xanthomonas

Production and localization of endogenous hydrogen peroxide (H₂O₂) were investigated in strains of Xanthomonas by histochemical analysis under electron microscopy. Even though the levels of endogenous H₂O₂ production were different among various strains, the produced H₂O₂ was localized in the cell wall of all Xanthomonas strains tested. The impairment of the level of endogenous H₂O₂ accumulation resulted in a significantly decreased growth rate of bacteria, regardless if the difference of the H₂O₂ level is originally present between wild type strains or caused by mutation of the ahpC gene of Xanthomonas. The endogenous accumulation of H₂O₂ positively correlates with the cell division. Interestingly, the accumulated H₂O₂ was also localized in the mesosome-like structure and nucleoids during the cell division cycle. Furthermore, results revealed quantitative and dimensional changes of H₂O₂ accumulation in the two additional locations. These findings indicated that the additional locations of the accumulated H₂O₂ were closely associated with the process of cell division. Together, these results suggest that the endogenous H₂O₂ production plays an important role in cell proliferation of Xanthomonas.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae)

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

Headspace ethylene accumulation effects on secondary metabolite production in Vaccinium pahalae cell culture

The influence of headspace ethylene on anthocyanin, anthocyanidin, and carotenoid accumulation was studied in suspension cultures of Vaccinium pahalae. Exogenous application of ethrel (an ethylene-releasing compound) significantly reduced growth and secondary metabolite production, whereas incorporation of 5.0 or 10.0 mg l^-1 CoCl₂ or NiCl₂ effectively reduced ethylene accumulation and improved product accumulation, but AgNO₃was toxic to cells. This study showed an overall negative impact of increased ethylene levels in the vessel headspace on phytochemical production in ohelo cell cultures.     

Effect of plant growth regulators and medium composition on cell growth and saponin production during cell-suspension culture of mountain ginseng (Panax ginseng C. A. mayer)

We have established cell-suspension cultures of mountain ginseng (Panax ginseng G A. Mayer), and have attempted to increase the yield of saponin by manipulating our processing method and culturing factors (e.g., media strengths; the presence of plant growth regulators or sucrose; ratios of NO⁺ ₃/ NH^- ₄). Maximum biomass yield was obtained in media containing 2,4-D. However, saponin productivity was much higher in a medium comprising either IBA or NAA; 7.0 mg/L IBA was optimal for promoting both cell growth (10.0 g/L dry weight) and saponin production (7.29 mg/g DW total ginsenoside). Although the addition of cytokinins (BA and kinetin) did not affect cell growth, the level of saponin (particularly in the Rb group) was enhanced when the media were supplemented with either 0.5 mg/L BA or 0.5 mg/L kinetin. Half- and full-strength MS media were equally suitable for inducing both biomass as well as saponin production. We also investigated the effect of various concentrations of sucrose and nitrogen, and found that 30 g/L sucrose enhanced biomass yield as well as saponin content However, further increases (i.e., up to 70 g/L) led to a decrease in saponin accumulation and biomass production. Maximum growth and saponin productivity were reported from treatments with an initial nitrogen concentration of 30 mM. In general, the amount of saponin increased when the test media had high NO⁺ ₃/ NH^- ₄ ratios; in fact, saponin production was greatest when nitrate was the sole nitrogen source.     

Efficient itaconic acid production by Aspergillus terreus: Overcoming the strong inhibitory effect of manganese

Itaconic acid (IA), a building block platform chemical, is produced industrially by Aspergillus terreus utilizing glucose. Lignocellulosic biomass can serve as a low cost source of sugars for IA production. However, the fungus could not produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate even at 100-fold dilution. Furfural, hydroxymethyl furfural and acetic acid were inhibitory, as is typical, but Mn²⁺ was particularly problematic for IA production. It was present in the hydrolyzate at a level that was 230 times over the inhibitory limit (50 ppb). Recently, it was found that PO₄³⁻ limitation decreased the inhibitory effect of Mn²⁺ on IA production. In the present study, a novel medium was developed for production of IA by varying PO₄³⁻, Fe³⁺ and Cu²⁺ concentrations using response surface methodology, which alleviated the strong inhibitory effect of Mn²⁺. The new medium contained 0.08 g KH₂PO₄, 3 g NH₄NO₃, 1 g MgSO₄·7H₂O, 5 g CaCl₂·2 H₂O, 0.83 mg FeCl₃·6H₂O, 8 mg ZnSO₄·7H₂O, and 45 mg CuSO₄·5H₂O per liter. The fungus was able to produce IA very well in the presence of Mn²⁺ up to 100 ppm in the medium. This medium will be extremely useful for IA production in the presence of Mn²⁺. This is the first report on the development of Mn²⁺ tolerant medium for IA production by A. terreus.     

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.