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Pseudomonas fluorescens as a physiological modulator in the enhancement of medicinally important alkaloids of Catharanthus roseus
Abstract
In the present investigation, changes in individual alkaloid profiles were studied in Catharanthus roseus (L.) G. Don. plants under treatment with Pseudomonas fluorescens. The Pseudomonas fluorescens treatments were given by soil drenching on 38, 53, 68 and 83 days after planting (DAP) by soil drenching. The plants were taken randomly on 45, 60, 75 and 90 DAP and used for estimating individual alkaloids like Ajmalicine, Catharanthine, Tabersonine, Serpentine and Vindoline contents. It was found that, Pseudomonas fluorescens has a profound effect and it caused a significant enhancement in the production of individual alkaloids when compared to untreated control plants.
... Bonilla et al. (2014) showed that the rhizosphere bacterium N5.18, Stenotrophomonas maltophilia, increased the total alkaloid yield of opium poppy. In addition to endophytic and rhizosphere bacteria, Jaleel et al. (2009) revealed that treatment with a low concentration of exogenous PGPR (P. fluorescens) increased the level of alkaloids in C. roseus. ...
... Additionally, Streptomyces inoculation was established to be highly advantageous compared with Bacillus for increasing alkaloid production. Presently, it has been revealed that microorganisms' amendment could be applied as a possible method to boost the concentration of main TIAs in periwinkle [87]. Chitosan is known to elicit activation, leading to a variety of defensive responses including accumulation of secondary metabolites [5]. ...
Periwinkle plant represents a major source of immensely vital terpenoid indole alkaloids and natural antioxidants which are widely used in cancer chemotherapy. A pot experiment was done to evaluate the role of two periwinkle endophytes (Streptomyces sp. and Bacillus sp.) with or without abiotic elicitors (aluminum chloride, tryptophan, and chitosan) on plant biomass, physio-biochemical attributes, phytopharmaceutical constituents, and alkaloid production. Inoculation with endophyte microbes significantly increased plant growth, nitrogen, phosphorus, potassium, carotenoids, ascorbic acid, and alkaloid yield. It also decreased oxidative biomarkers (hydrogen peroxide and malondialdehyde) and had no significant effects on flavonoids and anthocyanin. In this regard, Streptomyces sp. was more effective than Bacillus sp. Foliar spraying with chitosan significantly increased plant growth, chlorophyll, ions, antioxidant capacity, phytopharmaceutical constituents (total soluble phenols, flavonoids, and anthocyanin), and alkaloid yield, associated with a decline in oxidative biomarkers. Conversely, aluminum chloride application generally increased oxidative biomarkers, which was associated with a decreasing effect on plant growth, chlorophyll, and ions. Application of either tryptophan or chitosan with endophyte microbes increased plant growth, chlorophyll, ions, antioxidants, and alkaloid; meanwhile, it decreased oxidative biomarkers. On the contrary, aluminum chloride with endophytes evoked oxidative damage that was associated with a reduction in plant growth, chlorophyll, ions, and phytopharmaceutical constituents. The current study provides a proof-of-concept of the use of the endophyte Streptomyces sp. with chitosan for enhancing periwinkle plant biomass, phytopharmaceuticals accumulation, and alkaloid production.
... CATDLF5, CATDLF6, CATDRF2 and CATDS5, on the other hand, could enhance the serpentine (82Á5-337Á6%) content substantially. It has been noticed that exogenous Pseudomonas fluorescens application at low concentration can also enhance ajmalicine and serpentine content in plant root (Jaleel et al. 2009). The combined effect of arbuscular mycorrhizal fungi and nitrogen fertilizer in C. roseus has also been used for enhanced bioproduction of ajmalicine (Monnerat et al. 2018). ...
Aim:
The aim of study was to identify and characterize promising endophytes capable of enhancing the content of root alkaloids ajmalicine and serpentine in low alkaloid yielding genotype of C. roseus cultivar Prabal and the possible mechanisms involved.
Method and result:
Of the four strains isolated from alkaloid rich-genotype of C. roseus cultivar Dhawal, endophytic strains CATDLF5 (Curvularia sp.) and CATDLF6 (Choanephora infundibulifera) enhanced serpentine content by 211·7 - 337·6%, while CATDRF2 (Aspergillus japonicus) and CATDS5 (Pseudomonas sp.) increased the content of ajmalicine by 123·4 - 203·8% in cultivar Prabal. Upregulated expression of key genes, geraniol 10-hydroxylase, tryptophan decarboxylase and strictosidine synthase involved in terpenoid indole alkaloid biosynthetic pathway was observed in endophyte inoculated plants. Upregulated Octadecanoid-derivative Responsive Catharanthus AP2/ERF domain transcription activators like ORCA3 while, and downregulation of transcriptional repressor, ZCTs (Cys2/His2-type zinc finger protein family) enhanced the expression of genes for secondary metabolite production in endophyte inoculated plants.
Conclusion:
Present work concluded that the selected endophytes of C. roseus can enhance the ajmalicine and serpentine contents by modulating the expression of structural and regulatory genes of terpenoid indole alkaloid biosynthetic pathway in root.
Significance and impact of the study:
Endophytes can be play an important role to enhance in planta content of pharmaceutically important alkaloids in C. roseus and can therefore be useful in reducing the cost of production of important alkaloids.
... In an earlier study it has been shown that exogenous Pseu- domonas fluorescens application at low concentration could be used as a potential tool to increase the level of key terpenoid indole alka- loids in C. roseus (Jaleel et al., 2009). However, the V3 treatment in the present study provided a greater increase in content of key ter- penoid indole alkaloids (like vindoline, ajmalicine, and serpentine) as compared to the earlier study with P. fluorescens. ...
Catharanthus roseus is particularly known for its therapeutically useful terpenoid indole alkaloids, including the anticancer bisindole alkaloids (vinblastine and vincristine), as well as other alkaloids like ajmalicine and serpentine. The high cost, scarcity and commercially non-viable chemical syntheses of the bisindole alkaloids have sustained the research effort on the plant till date. The present study was undertaken to determine the effect of endophytes isolated from C. roseus on the growth parameters/biomass and in planta content of key terpenoid indole alkaloids like vindoline, vincristine, serpentine and ajmalicine. Out of the four endophytes isolated three were bacterial (V1, V2, V3) and one was fungal (V4). In the initial glasshouse experiments, V1 and V3 treatments were found to outperform the remaining two in terms of improvement in plant growth parameters. The bacterial endophytes, V1 and V3 were identified as Staphylococcus sciuri and Micrococcus sp., respectively through 16S rRNA gene sequence analysis. Treatment with both these endophytes was used on field grown plants to determine their effect on plant growth parameters and in planta contents of selected terpenoid indole alkaloids. It was found that both of them significantly increased the plant height but V3 was found to be superior to V1 when enhancement in weight of shoot, root and leaf or in planta content of vindoline, ajmalicine and serpentine was considered. The results of this study clearly indicate the possibility of using bacterial endophytes like V3 as bio-inoculants for enhancing plant growth/biomass and in planta content of key terpenoid indole alkaloids like vindoline (that may be used for bisindole alkaloid semi-synthesis), serpentine and ajmalicine.
Endophytes play a vital role in the survival of host plants by aiding defense responses by producing bioactive compounds similar to their host plants. To fulfill the ever-increasing demand for herbal drugs to cure human ailments, researchers are searching for the various sources of bioactive compounds besides medicinal plants. It was reported that in the international market, the demand of 3 kg per year of vinca alkaloids (1.5×106 kg dry leaves are required) to develop powerful plant-derived anticancer drugs. In this regard, this review aims to highlight the endophytes residing in Catharanthus roseus (family: Apocynaceae), capable of synthesizing indole alkaloids, vinblastine, vincristine, vindoline, vinflunine, vincamine, ajmalicine, ajmaline, serpentine, and reserpine used to control cancer, diabetes, malaria, vascular dementia, cardiac diseases, etc. In the search to fulfill the demand, there is urgent need to develop an efficient method of isolation, identification of endophytes, and the down-streaming process for more efficient and sustainable production of vinca alkaloids from endophytic fungus for the cancer treatment products. Microbial fermentation by optimizing media composition, precursors, inducers, and the metabolic bypass inhibitors would be a promising method in the production of vinca alkaloids at industrial scale. Furthermore, different biotechnological strategies such as gene cloning, gene transformation, and mutations can widely be used on endophytic fungi and bacteria in order to increase the productivity of the vinca alkaloids. Thus, advancements in science and technology have increased the extraction yield from vinca alkaloid producing endophytes, thereby improving the overall efficiency of alkaloid production.
Key message
This paper showed that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.
Abstract
The aim of this study was to investigate the relationship between nitric oxide (NO) and polyamines (PAs) and to determine their roles in betulin accumulation induced by the endophytic fungus Phomopsis in Betula platyphylla. Treatment of birch plantlets with the endophytic fungus Phomopsis promoted an NO burst and accumulation of PAs and betulin. Birch plantlets were treated with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the PA synthesis inhibitor d-arginine (d-arg). cPTIO and d-arg inhibited the fungus-induced NO burst and accumulation of PAs and betulin. The exogenous NO donor sodium nitroprusside promoted PA production and betulin accumulation, whereas an exogenous PA, putrescine, promoted an NO burst and betulin accumulation. In addition, d-arg inhibited NO production and cPTIO decreased PA production during fungus-induced betulin accumulation. Our results indicate that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.
Catharanthus roseus poses a wide range of medicinally important alkaloids such as Vincristine and Vinblastine that are used in the treatment of cancer. The other alkaloids such as Ajmalicine and Serpentine are used as sedative compounds. Industrial production of these alkaloids, extremely exploits Catharanthus roseus, however, tissue culture is an alternative approach but low yield is a major problem. Elicitor treatment is an effective strategy used in improvement of production of alkaloids. Both biogenic, abiogenic and their combinations showed some promising results. Concentration of elicitors and application time is crucial phenomenon in production of different alkaloids.
A field study was conducted to evaluate the effectiveness of arbuscular mycorrhizal fungi
(AMF) and phosphorus levels (100, 150 and 200 kg) for increasing biomass yield and
ajmalicine content in a medicinal plant (Catharanthus roseus). The plants treated with 150 and
200 kg P2O5/ha along with AMF had the maximum plant height, number of leaves, root
biomass, phosphorus content, root colonization, spore count and ajmalicine content 120 days
after planting when compared with the control plants. The results suggested that these
treatments could be recommended for enhancing biomass and alkaloid content in C. roseus.
AIM: To evaluate the antioxidant potential and indole alkaloid variations in different parts of two varieties (rosea and alba) of Catharanthus roseus. METHODS: The antioxidant potentials were examined in terms of non-enzymatic antioxidant molecules and activities of antioxidant enzymes. The non-enzymatic antioxidant molecules studied were ascorbic acid, α-tocopherol and reduced glutathione. The estimated antioxidant enzymes were superoxide dismutase, ascorbate peroxidase, catalase, peroxidase and polyphenol oxidase. The analyses were carried out in the field-collected samples. The antioxidant and alkaloid profiles were estimated from roots, stems, leaves, flowers and pods. RESULTS: The roots and stems had higher antioxidant content when compared with other parts of the plant. The lowest content was found in flowers and pods. The rosea variety was good performer in terms of antioxidant and alkaloid contents. The activities of antioxidant enzymes were higher in leaves when compared with other parts. But indole alkaloid content was found to be higher in the roots of rosea variety. CONCLUSION: The pink-flowered rosea variety of Catharanthus roseus is more suitable for cultivation in terms of antioxidant and alkaloid contents.
An investigation was carried out with paclobutrazol (PBZ) in order to study its effect on the photosynthetic and anatomical characteristics of Catharanthus roseus (L.) G. Don. The chlorophyll content and related parameters like net photosynthetic rate, transpiration rate and internal CO2 concentration were measured from control and treated plant samples. The anatomical characteristics, viz., leaf thickness, epidermis and cuticle, palisade layer, spongy layer and vascular bundle were studied. The root alkaloid ajmalicine was extracted and estimated from both treated and control plant roots. Photosynthetic pigment increased under PBZ treatment. PBZ treatment increased the net photosynthetic rate and internal CO2 concentration, but reduced transpiration. PBZ treatment increased the thickness of the leaf, epidermis and cuticle, palisade layer and spongy layer but reduced the diameter of xylem vessels significantly when compared to control, however the phloem elements had shown an increased diameter as compared to control in the PBZ treated plants. There was a significant enhancement in ajmalicine content under PBZ treatment when compared to control plants. The results suggested that the application of PBZ have significant effects on photosynthetic and anatomical responses thus can be used for improving productivity in medicinal plants.
The ability of triadimefon (TDM), a triazole group of fungicide, to ameliorate salinity stress was studied in Catharanthus roseus (L.) G. Don. plants subjected to sodium chloride (NaCl) treatment. NaCl treatment at 80mM level decreased overall growth
of this plant and reduced the chlorophyll contents, protein, antioxidant enzymes such as peroxidase (POX), superoxide dismutase
(SOD) and polyphenol oxidase (PPO). The root alkaloid ajmalicine got increased under salt treatment. When these stressed plants
were treated with TDM at 15mgl−1 concentration minimized the inhibitory effects of NaCl stress by increasing the root, shoot growth and leaf area and increased
dry weight (DW), chlorophyll, protein contents and the activities of antioxidant enzymes like POX, SOD and PPO, thereby paved
the way to overcome the salinity injury. The quantity of ajmalicine was again increased with the TDM treatment when compared
to both control and NaCl treated plants. From these results, it is proved that the fungicide TDM have great role in the enhancement
of plant antioxidative enzymes and the enhanced scavenging of potentially harmful free radicals, as a mechanism of protecting
plants against noxious oxidative stress from the environment and also in the enhancement of active principles.
An investigation was carried out to estimate the salt stress ameliorative effect of propiconazole (a triazole group of fungicide cum plant growth regulator) on the NaCl stressed Catharanthus roseus. The experiment was conducted in pots. All the pots were irrigated to the field capacity with ground water up to 30 days after sowing (DAS). The treatments were given as 80 mM NaCl, 80 mM NaCl + 20 mg l−1 propiconazole and 20 mg l−1 propiconazole alone on 30, 50 and 70 DAS. The samples were collected on 60 and 80 DAS. NaCl stress inhibited the root and stem length, total leaf area, fresh and dry weight, and antioxidant enzymes superoxide dismutase (SOD), peroxidase (POX) and polyphenol oxidase (PPO) activities. Plants treated with NaCl with propiconazole increased these parameters to a larger extent when compared to NaCl stressed plants. The propiconazole treatment increased the root length, fresh and dry weight, and SOD, POX and PPO activities, but decreased the stem length and leaf area when compared to control. The results showed that the propiconazole could significantly ameliorate the effect of NaCl stress in Catharanthus roseus.
The effect of different plant growth regulators (PGR) and elicitor treatments on the alkaloid profile variation of Catharanthus roseus was investigated in the present study. The PGR used were paclobutrazol (PBZ), gibberellic acid (GA3) and Pseudomonas fluorescens elicitors (PF Elicitors). The estimated alkaloids were ajmalicine, catharanthine, tabersonine, serpentine and vindoline. In roots, the ajmalicine content increased significantly under all the treatments on all sampling days. In roots, the catharanthine contents increased with the age in control and growth regulator treatments, but the increase was not prominent and significant in PGR treatments when compared to controls. The serpentine contents of the plant increased with PGR treatments, but the increase was more prominent in PBZ treatments when compared to other treatments. The increase was in the order PBZ > PF Elicitors > GA3. C. roseus never showed any significant increase in tabersonine contents in the roots under GA3 treatments, but it increased significantly under PBZ and PF Elicitors when compared to control plants. The root vindoline contents increased with PBZ and PF Elicitors treatments but the decreased under GA3 treatments when compared to control plants. Our results have good significance, as these increases the secondary metabolites of this traditional medicinal plant.
The effect of triadimefon was investigated in a medicinal plant, Catharanthus roseus subjected to water deficit stress. The abscisic acid (ABA) level, DNA and RNA contents and activities of ATPase and protease were found varying in different parts of the plants under treatment. Drought treatment increased the ABA level more than twofold in all parts of the plants. TDM treatment to the drought stressed plants showed highest contents. In roots, stem and leaves, drought stress caused a decrease in the DNA and RNA contents when compared with control and other treatments. TDM treatment with drought increased the nucleic acid contents to the level of the control roots. The activity of ATPase and protease were increased under drought treatment and lowered due to TDM applications. This information could be useful in the field of soil water deficits reclamation efforts by using plant growth regulators.
Changes in antioxidant potentials and indole alkaloid, ajmalicine, production were studied in Catharanthus roseus (L.) G. Don. plants under treatment with gibberellic acid (GA(3)). The GA(3) treatments were given in two ways, foliar spray and soil drenching methods on 30, 45, 60 and 75 days after planting (DAP). The plants were uprooted randomly on 90 DAP and separated into root, stem and leaves and used for analyses. The antioxidant potential was studied in terms of non-enzymatc antioxidant molecules like ascorbic acid (AA), alpha-tocopherol (alpha-toc) and reduced glutathione (GSH) and activities of antioxidant enzyme, viz., superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT). The alkaloid ajmalicine was extracted and estimated from roots of both control and treated plants. It was found that, GA(3) has a profound effect upon the antioxidant potentials and it caused a significant enhancement in the production of ajmalicine when compared to untreated control as well as foliar-sprayed plants. There was no significant enhancement in GSH and ajmalicine content under GA(3) foliar spray in C. roseus. These preliminary results suggest that, the application of GA(3) may be a useful tool to increase the antioxidant potential and alkaloid production in medicinal plants like C. roseus.
Triadimefon (TDM), a systemic fungicide with non-traditional plant-growth regulator properties, was administered to Catharanthus roseus (L.) G. Don. plants in order to determine its effects on oxidative injury in terms of H2O2 content, lipid peroxidation (LPO), electrolyte leakage (EL), protein and amino acid contents, as well as proline metabolism. The LPO, estimated as thiobarbituric acid-reactive substances (TBARS), decreased under TDM treatment. It was found that H2O2 and EL were reduced under TDM treatment when compared to control. TDM treatment caused a significant increase in the protein and amino acid contents. Glycine betaine (GB) and proline (PRO) significantly accumulated in C. roseus under stress arisen from fungicide applications. Proline oxidase (PROX) activities reduce the PRO content and gamma-glutamyl kinase (gamma-GK) accelerates the synthesis of PRO. Under TDM treatment, the activity of PROX decreased and the gamma-GK activity increased. From our results, it is suggested that fungicide triadimefon causes activation of metabolic processes in the medicinal plant Catharanthus roseus. These findings are of great significance for the cultivation of this medicinal plant, as it was previously reported that TDM causes an enhancement of antioxidant metabolism and ajmalicine production in C. roseus.
Triadimefon, potential fungicide cum plant-growth retardant was used in this study to investigate its effect on the growth and the photosynthetic pigment contents of two varieties of Catharanthus roseus (L.) G. Don. The plants of both varieties were subjected to 15 mg l(-1) triadimefon treatment by soil drenching 30, 45, 60, and 75 days after planting (DAP). Plants were uprooted on 90 DAP, and morphological parameters, like plant height, number of leaves, leaf area, root length and fresh and dry weights were determined. The photosynthetic pigments, like chlorophylls a and b, total chlorophyll, carotenoids, floral pigment, anthocyanin, were extracted and estimated. It was observed that plant height, number of leaves and leaf area were decreased and that root length, fresh and dry weights were increased under triadimefon treatment. The photosynthetic and floral pigments were increased under triadimefon treatment in both varieties. The results suggest that the application of this plant-growth retardant (triadimefon) has favourable effects on the reduction of plant height; it can thus be used for replacing manual hand pruning and for improving floral and vegetation colour in bedding plants like C. roseus.