Jasmonic acid elicitation of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones

Hypericum perforatum L. cell suspensions were evaluated for their viability, growth, dark gland formation and ability to produce phenylpropanoids and naphtodiantrones after elicitation with different jasmonic acid (JA) concentrations. Phenolic compounds were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization mass spectrometry (ESI-MS). The activities of two key enzymes of the phenylpropanoid/flavonoid pathways, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also monitored to estimate general channeling in the different metabolic pathways. A 6-fold increase of phenolic compounds, flavanols and flavonols after JA elicitation was observed in cells. In contrast, anthocyanins were in lower amounts in JA treated cells suggesting a modification of the channeling in the phenylpropanoid pathway. Similar accumulations with maxima after 4 days of elicitation were found for naphtodianthrones (2.4-fold) such as hypericin and pseudohypericin in cells. At least a 6–8-fold increase of PAL and CHI activities was observed in JA elicited cells confirming a strong activation of the phenylpropanoid pathway. JA elicitation increased production of phenylpropanoids and naphtodianthrones in H. perforatum cell suspension without differentiation of dark glands under 16 h photoperiod.     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

Significantly improved taxuyunnanine C production in cell suspension cultures of <Emphasis Type="Italic">Taxus chinensis</Emphasis> by process intensification of repeated elicitation,sucrose feeding,and in situ adsorption

A highly efficient process intensification strategy was developed to enhance the taxuyunnanine C (Tc) production in Taxus chinensis (T. chinensis) by combining repeated elicitation with a synthesized jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA), sucrose feeding and in situ adsorption. Suspension cultures of a high Tc-producing cell line of T. chinensis were supplemented twice, on day 7 and day 12, with 100 μM DHPJA. The cultures were also supplemented by feeding 20 g sucrose/l and 100 g Ambelite XAD-7/l on day 7. This strategy had a synergistic effect on Tc synthesis, causing a significant increase in the Tc yield to 1,715.13 ± 124.12 mg/l on day 21. The yield was 3.28-fold above that produced by repeated DHPJA elicitation combined with sucrose feeding. When XAD-7 was replaced by Amberlite XAD-7 HP, the result was slightly different. The highest Tc yield of 1,728.84 ± 50.37 mg/l was achieved on day 27. The Tc yield achieved here is greater than twice the highest yield reported previously, the sole reported taxoid production yield above the gram per liter level. Given the pharmaceutical importance of taxoids, the results of this study will greatly contribute to the potential industrial production of Tc and other taxoids by plant cell cultures.     

An elicitor-and pathogen-induced cdna from potato encodes a stress-responsive cyclophilin

Differential mRNA display was used to identify pathogen-responsive, stress-related genes in potato cell suspensions treated with salicylic acid and a cell wall-derived elicitor from Phytophthora infestans. Among the positive clones identified, one was found to be expressed at a significantly higher level in elicited cells than in control cells. DNA sequencing of this amplicon revealed high homology and identified it as a potato cyclophilin cDNA. The maximum amount of the cyclophilin mRNA was found 9 to 12 h after elicitation. Cyclophilin (CyP) mRNA synthesis was also up-regulated from 12 to 24 h in potato leaves locally infected with zoospores from Phytophthora infestans. However, untreated leaves responding systemically to the pathogen showed only a weak, delayed response at 24 h post infection. The observed accumulation of potato CyP mRNA in response to salicylic acid, P. infestans elicitor and P. infestans infection, suggest that CyPs play an important role in plant stress responses.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.     

Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.     

Role of antibodies and effect of BCG vaccination in experimental candidiasis in mice

The role of humoral antibodies and the effect of BCG vaccination were studied in the experimental candidiasis in mice. The antibody suppressed, B-cell deficient animals were prepared by repeated administration of rabbit anti-mouse--antiserum to the new born mice from birth onwards. Such immunodeficient animals along with controls were infected intravenously with Candida albicans, to study the course of candidal infection. It was observed that B-cell-deficient animals were found to be more susceptible to candidal infection than the controls, as indicated by their steady loss of body weight, longer mean time to death and higher viable counts of candidal cells in different organs. The anti-candidal antibodies were absent in all B-cell-deficient animals but present in the controls. These results suggest that antibodies make a contribution in protection against candidal infection in mice. The BCG vaccinated animals were prepared by repeated intravenous administration of BCG to mice and these vaccinated animals along with unvaccinated controls were challenged intravenously with C. albicans, to study the course of candidal infection. It was observed that BCG vaccination prolonged meantime to death and reduced the number of candidal cells in their kidneys.     

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.     

Patterns of protein expression upon adding sugar and elicitor to the cell culture of Eschscholtzia californica

The optimal timing of elicitation was determined for the production of benzophenanthridine alkaloids (BPAs) by Eschscholtzia californica cell culture. Upon elicitation, 7-day old cells produced more alkaloids than 14-day old cells (5.1 times for sanguinarine and 2.7 times for dihydrosanguinarine). We presumed that these alkaloids are growth-rate-associated secondary metabolites in E. californica cell culture. Although the specific productivity of alkaloids were higher in 7-day old culture, the total cell mass of 7-day old culture was about half that of 14-day old culture. In order to increase the overall productivity, sucrose was added to the 14-day old culture before the addition of elicitor. By this way, cells in the stationary phase (14-day old culture) could be switched to the cells in the logarithmic growth phase (similar to 7-day old culture). Total production of alkaloids was increased by adding sucrose; especially the production of sanguinarine was increased as high as 5.7 times of the control. To find out the protein level changed by the elicitation, proteins extracted from whole cell were separated by using two-dimensional gel electrophoresis. The patterns of the gels were different and little correlation among the proteins could be observed. And Western blotting was employed to check the expression level of selected five enzymes, these enzymes believed to be involved in BPAs production, resulting in up-regulated with elicitor addition.     

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.     

Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal

An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.     

Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation

A single addition of 200 M methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume). Repeated elicitation with two additions of 200 M MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels. The content of ginsenosides Rg₁, Re, Rb₁ and Rd in the ALR was increased from, respectively, 0.18±0.01, 0.21±0.01, 0.21±0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32±0.02, 0.36±0.02, 0.72±0.06 and 0.08±0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43±0.02, 0.46±0.03, 1.09±0.07 and 0.14±0.02 mg per100 mg DW with two additions of MJA. Interestingly, the activity of the Rb₁ biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb₁ content. In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted. The final cell density and total ginsenoside content in the ALR reached 27.3±1.5 g/l and 2.02±0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg₁, Re, Rb₁ and Rd was 111.8±4.7, 117.2±4.6, 290.2±5.1 and 32.7±8.1 mg/l, respectively. The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.     

Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hydroxyethyl jasmonate

Chemically synthesized 2-hydroxyethyl jasmonate (HEJA) was for the first time employed to induce the ginsenoside biosynthesis and to manipulate the product heterogeneity in plant cell cultures. The dose response and timing of HEJA elicitation were investigated in cell suspension cultures of Panax notoginseng. The optimal concentration and timing of HEJA addition for both cell growth and ginsenoside accumulation was identified to be 200 μM added on day 4. It was interestingly found that HEJA could stimulate ginsenosides biosynthesis and change their heterogeneity more efficiently than methyl jasmonate (MJA), i.e., the total ginsenoside content and the Rb/Rg ratio increased about 60 and 30% with HEJA elicitation than that by MJA, respectively. The activity of Rb₁ biosynthetic enzyme, i.e., UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), was also higher in the former case. A maximal production titer of ginsenoside Rg₁, Re, Rb₁, and Rd was 47.4±4.8, 52.3±4.4, 190±18, and 12.1±2.5 mg/l with HEJA elicitation, which was about 1.3-, 1.3-, 1.7-, and 2.1-fold than that using MJA, respectively. Early signal events in plant defense response, including oxidative burst and jasmonic acid (JA) biosynthesis, were also examined. Levels of H₂O₂ and NO in medium and l-phenylalanine ammonia lyase activity in cells were not affected by addition of MJA and HEJA. On the other hand, the JA content in cells was increased with external jasmonates elicitation, and it was inhibited with the addition of JA biosynthesis inhibitors. The results suggest that oxidative burst might not be involved in the jasmonates-elicited signal transduction pathway, and MJA and HEJA may induce the ginsenoside biosynthesis via induction of endogenous JA biosynthesis and key enzymes (such as UGRdGT) in the ginsenoside biosynthetic pathway of P. notoginseng cells. The information is useful for hyperproduction of plant-specific heterogeneous products.     

Defense responses elicited in pine cell suspension cultures

To understand mechanisms of disease resistance in pine trees, we took advantage of the fact that suspension cultured cells exhibit many of the defense responses that are characteristic of intact tissues. In this study, we measured constitutive and elicitor-induced levels of ethylene production, chitinase activity and glucanase activity in cells of loblolly pine (Pinus taeda L). Increased ethylene production was induced similarly by a live fungus (Ophiostoma minus Hedgc. H.P. Sydow) and chitosan, a general elicitor. Culture age, relative to the most recent transfer, affected the constitutive level of all defense responses. Culture age also had a pronounced effect on the ability of the cells to produce ethylene and cellular chitinase, but not on secreted chitinase, cellular glucanase, secreted glucanase, or lignification. In older cultures, elicitation induced a 4- to 10-fold increase in ethylene production and a 2-fold increase in cellular chitinase, secreted chitinase and cellular glucanase. Chitosan elicitation did not affect secreted glucanase. The overall regulation of the defense response in pine cells appears complex, but individual components of the response can be differentially induced in cell cultures under appropriate experimental conditions.     

Growth Characteristics and Elicitor-induced Reactions of Photosynthetically Active and Heterotrophic Cell Suspension Cultures of Lycopersicon peruvianum (Mill.)*

Cell suspension cultures of Lycopersicon peruvianum (Solanaceae) were established as well-growing photoautotrophic, photomixotrophic and heterotrophic cultures and their growth parameters were characterized. Elicitor-induced responses of these cultures to the tomato pathogen Fusarium oxysporum f. sp. lycopersici were investigated after treatment of cells with autoclaved mycelium and culture filtrate of this fungus. The dominant reaction was an enhanced incorporation of phenolic constituents in the plant cell wall. Among the nine phenolics released by alkaline hydrolysis the most prominent compounds were p-hydroxybenzaldchyde, vanillin, p-coumaroyltryamine, feruloyltyramine, p-coumaric acid and ferulic acid. Phenolic incorporation in cell walls resulted in increased stability of cells against protoplasting with microbial enzymes. Chlorogenic acid, as the main soluble phenolic compound, showed differential accumulation in the three cell cultures lines as well as an elicitor-induced transient decrease. In heterotrophic cells decrease of chlorogenate occurred concomitant with accumulation of caffeoyl- and p-coumaroylshikimate as well as increased activities of p-coumaroyleoenzyme A: shikimic acid p-coumaroyltransferase. Upon elicitation increased activities of phenylalanine ammonia lyase and changes in peroxidase activities wore also detected. Sesquiterpenoid phytoalexines were not produced by either one of the cell culture lines and levels of tomatine were not significantly affected by elicitation.     

Chitinase in cucumber hypocotyls is induced by germinating fungal spores and by fungal elicitor in synergism with inducers of acquired resistance

After root pretreatment with 2,6-dichloroisonicotinic acid (DCIA or INA), hypocotyls of etiolated cucumber seedlings acquired resistance to infection by Colletotrichum lagenarium caused by the failure of the fungus to penetrate epidermal cell walls. The hypocotyls contained only low levels of class III chitinase and its mRNA prior to infection. This pathogenesis-related (PR) gene was expressed strongly upon infection but only in resistant hypocotyls and soon after germination of the fungal spores. Chitinase was also induced early by an albino mutant strain of C. lagenarium that can barely penetrate the epidermis. Thus, early recognition of the fungus implies signal compounds able to pass, or being generated in, the hydrophobic epidermal surface. As the apoplastic chitinase accumulates timely at the site of a subsequent attack, it may contribute to disease resistance. The mechanism behind the enhanced responsiveness of epidermal cells was studied by gently abrading the cuticle of susceptible hypocotyls to allow permeation of a water-soluble polymeric fungal elicitor. Induction of chitinase occurred only when the elicitor was applied simultaneously with a resistance inducer such as DCIA, salicylic acid (SA) or a benzothiadiazole (BTH). In addition, long-term root pretreatment with DCIA conditioned the hypocotyls for enhanced elicitor responses. These results demonstrate that the above inducers of acquired resistance can affect expression of the cucumber chitinase gene not only as direct inducers. They can also act synergistically with fungal elicitors and, in addition, condition the hypocotyls in a developmental manner for potentiated elicitation.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.