Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor

AIMS: To determine the role of fungal metabolites in the desorption of metals. METHODS AND RESULTS: Desorption of Zn from charcoal by three different fungi was compared against metal desorption with reverse osmosis water, a 0.1% Tween 80 solution and a 0.1 mol l(-1) CaCl(2) solution. All three fungal filtrates desorbed three times more Zn than either 0.1% Tween 80 or 0.1 mol l(-1) CaCl(2). Metal chelator production in Trichoderma harzianum and Coriolus versicolor was constitutively expressed while chelator production in Trichoderma reesei was induced by Zn. The presence of Zn inhibited the production of metal chelators by C. versicolor. Only C. versicolor was found to produce oxalic acid (a strong metal chelator). All fungi caused a marked decrease in pH, although this was not enough to explain the increased desorption of the metals by the different fungal filtrates. CONCLUSIONS: Metal chelation via organic acids and proteins are the main mechanisms by which the fungal filtrates increase zinc desorption. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study explain why plants inoculated with T. harzianum T22 take up more metal from soil, than noninoculated plants while metabolites produced by fungi could be used for metal leaching from contaminated soils.     

Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus

Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a 'three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a beta-glucuronidase-green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed.     

Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture

Sugar-limited batch growth of Dioscorea deltoidea and Catharanthus roseus plant cell cultures was studied in a 14-L stirred tank fermentor. With dissolved oxygen concentration monitored and maintained at nonlimiting levels, growth rates and ratios of dry weight to fresh weight were found to be strongly influenced by sugar concentration. Linear relationships between respiration rate and growth rate were observed, and respiration rate was found to drop to a maintenance level after sugar had been fully depleted from the medium. Diosgenin biosynthesis by D. deltoidea was shown to be independent of growth rate. Ajmalicine biosynthesis in C. roseus was negligible during sugar-limited growth but was induced by inoculation into an 80-g/L glucose solution.     

Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus

A synchronous cell division system was established using the double phosphate starvation method, based on the observation that one of the limiting factors in the growth of a suspension culture of Catharanthus roseus (L.) G. Don cells in the medium of Murashige and Skoog was phosphate. In the system, an increase in cell number took place in a short period of only 4 h, while the cell number remained almost constant during other periods of the cell cycle. The synchrony of the culture was confirmed by changes in mitotic index, which increased sharply prior to the increase in cell number. The S phase was determined by measuring incorporation of [³H]-thymidine into the DNA fraction during the cell cycle and synchrony of DNA synthesis was verified likewise. Synchronization by phosphate starvation is discussed in relation to the function of phosphate as a nutrient. The synchronous system thus established will be useful in biochemical studies of the cell cycle in higher plants.     

Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus plants

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.     

Growth and stoichiometry of a Catharanthus roseus cell suspension culture grown under nitrogen-limiting conditions

The uptake of carbohydrate and nitrate by Catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. Biomass production was similar when using either 6, 12, 18, or 24 mM nitrate as the nitrogen source and 20 g L(-1) sucrose as the carbon source. In all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. Apparent biomass yields, Y(X/S) and Y(X/N) were 0.49 g biomass g(-1) glucose equivalent and 0.23 g biomass mmol(-1) nitrate, respectively. The determination of the cellular carbon-to-nitrogen ration (C/N ration) resulted in the identification of three district growth phases: an active growth phase, and accumulation phase, and a biomass decline phase (endogenous metabolism). The onset of the last two phases was correlated with nitrate and sugar of the last two phases was correlated with nitrate and sugar exhaustion, respectively. Balanced stoichiometric equations describing the active growth and accumulation phases were proposed based on elemental composition and ash content of the biomass. The stoichiometric equation related to the accumulation phase predicts that the available sugars are stored as starch- and lipid-like materials.     

Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions

Summary A system for the continuous cultivation of plant cells has been developed, based on a commercially available 3–1 turbine-stirred fermentor. A special device was constructed to provide for homogeneous effluent from the culture at low dilution rates. Two steady states with Catharanthus roseus cells growing under glucose limitation are described with respect to biomass yield on the carbon and energy source glucose, specific oxygen consumption, specific carbon dioxide production and (by)product formation. From a carbon balance for each steady state it is shown that the flow of carbon to the culture (as glucose) practically equalled the flow of carbon from the culture (as biomass, carbon dioxide and (by)product). Biomass yields on glucose were 0.31 g/g and 0.35 g/g at dilution rates of 0.0060 l/h and 0.0081 l/h respectively. The striking difference between the obtained yield coefficients and biomass yield commonly found for batch-cultured plant cells is discussed.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.     

长春花愈伤组织的诱导及限速酶活性的研究

长春花生物碱是目前应用最有效的天然植物抗肿瘤药物,目前利用生物工程生产长春花生物碱成果甚微。通过对长春花愈伤组织的诱导条件进行研究,同时对植株各部位的色氨酸脱羧酶(TDC)、过氧化物酶(POD)进行精确测定。认为长春花愈伤组织的诱导以MS培养基、激素2,4-D 2 mg/L和6-BA 0.5 mg/L最好,生物碱的合成是植物体多器官共同参与的结果。     

Two-phase culture for enhanced alkaloid synthesis and release in a new airlift reactor by Catharanthus roseus

A new airlift reactor was used to culture Catharanthus roseus cells, in which the draft tube was made up of polyurethane foam and acted as the immobilizing matrix. The reactor was connected in series to an adsorbent column with a neutral polymeric resin which absorbs these alkaloids. The synthesis of alkaloid was stimulated by adding the resin column and the total content of alkaloid secreted by cells reached 380 mg/L, which was 4.5 times of that in the control experiment. Meanwhile, most of the intracellular alkaloid produced by Catharanthus roseus was secreted into the medium.     

Changes in intracellular UDP-sugar levels during the cell cycle in a synchronous culture of Catharanthus roseus

UDP-sugar contents were measured using high performance liquid chromatography and gas chromatography during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-xylose and UDP-arabinose could be determined, and 75–90% of the UDP-sugars were UDP-glucose. The contents of UDP-glucose and UDP-galactose increased in the late G2-M and the late S-M phases, respectively, whereas UDP-glucoronic acid and UDP-arabinose increased in amount in the G1 phase. These changes in the levels of UDP-sugars during the cell cycle generally correlated well with the changes in cell wall constituents and in the activities of the enzyme involved in synthesis and interconversion of UDP-sugars reported by S. Amino et al. (Physiol. Plant. 1985. 64: 111–117).     

Changes in cell wall constituents during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in cell wall constituents during the cell cycle were investigated using a synchronous culture of Catharanthus roseus (L.) G. Don which was obtained by the double phosphate starvation method (S. Amino et al. 1983. Physiol. Plant. 59: 393–396). Cell walls isolated from the cells in each phase of the cell cycle were fractionated into EDTA-soluble (pectin), 5 and 24% KOH-soluble (hemicellulose) and 24% KOH-insoluble (cellulose) fractions. Their sugar compositions were investigated by gas chromatography and methylation analysis. The following changes were observed: (1) a significant increase in total cell walls in the G1 phase after cell division, (2) a temporary increase in the relative amount of the EDTA-soluble fraction during cytokinesis, (3) an increase in the relative amount of galactose, probably 4-linked galactose, in the EDTA-soluble fraction prior to cytokinesis, (4) a temporary increase in the relative amount of 3-linked glucose during cytokinesis, (5) little change in the composition of polysaccharides throughout the cell cycle in the 24% KOH-soluble fraction, which consisted mainly of xyloglucan. The changes observed are discussed in relation to the progression and physiological significance of each phase of the cell cycle.     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

Molecular cloning and characterization of Crmdr1, a novel MDR-type ABC transporter gene from Catharanthus roseus.

A novel gene encoding a MDR-like ABC transporter protein was cloned from Catharanthus roseus, a medicinal plant with more than 120 kinds of secondary metabolites, through rapid amplification of cDNA ends (RACE). This gene (named as Crmdr1; GenBank accession no.: DQ660356) had a total length of 4395 bp with an open reading frame of 3801 bp, and encoded a predicted polypeptide of 1266 amino acids with a molecular weight of 137.1 kDa. The CrMDR1 protein shared 59.8, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAD31576), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Crmdr1 was a low-copy gene. Expression pattern analysis revealed that Crmdr1 constitutively expressed in the root, stem and leaf, but with lower expression in leaf. The domains analysis showed that CrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that CrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.     

Effect of antimicrobial activity of Melaleuca alternifolia essential oil on antagonistic potential of Pleurotus species against Trichoderma harzianum in dual culture

Melaleuca alternifolia (tea tree) essential oil was investigated for its “in vitro” ability to control Trichoderma harzianum, a fungal contaminant that causes extensive losses in the cultivation of Pleurotus species. The antifungal activity of M. alternifolia essential oil and antagonist activities between Pleurotus species against three T. harzianum strains were studied in dual-culture experiments on an agar-based medium in which different concentrations of essential oil were incorporated. M. alternifolia essential oil at a concentration of 0.625 μL/mL, inhibited T. harzianum mycelial growth by 5.9–9.0%, depending on the strain. At the same concentrations P. ferulae and P. nebrodensis stimulated mycelial growth by 5.2–8.1%. All strains of T. harzianum were antagonistic to the Pleurotus species in the control. When essential oil was added to the substrate cultural, the antagonistic activity of T. harzianum against the Pleurotus species was weak (0.0625 μL of essential oil) or non-existent (0.125 μL of essential oil). M. alternifolia essential oil could be an alternative to the synthetic chemicals that are currently used to prevent and control T. harzianum in mushroom cultivation.     

BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

A new component of the beta-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.     

Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture

Here the current status of knowledge on some well-characterized transporters located in the vacuolar membrane is reviewed. As different cellular compartments and even different cells may be involved in certain steps of a biosynthetic pathway, the regulation of the flux is not only dependent on structural genes encoding enzymes catabolizing certain steps but also transport has a major regulatory function. The aim of the present review is to give an overview of the present knowledge of transport of secondary metabolites in plants, and to use this information in the context of our knowledge about Catharanthus roseus alkaloid biosynthesis. This should lead to further insight in the possible role of various transporters in the regulation of the biosynthesis of these alkaloids.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Isolation of Vinblastine in Callus Culture with Differentiated Roots of Catharanthus roseus (L). G. Don

We expressed and purified an azoreductase homolog, YvaB, from Bacillus subtilis. YvaB was found to have NADH:2,6-dichloroindophenol oxidoreductase activity, as well as azoreductase activity. Purified YvaB was active without FMN, unlike Escherichia coli azoreductase. YvaB was most active at pH 7.5 and 40 °C, and was stable up to 55 °C after incubation for 30 min. Remarkably, it was stable in the presence of Ag⁺, and was activated by the addition of non-ionic detergents. Other enzymatic properties of YvaB were also investigated.