Metabolites In Plant Cell Cultures Research Articles

Plant Tissue Culture and Metabolite Profiling for High-Value Natural Product Synthesis.

The engineering of plant cell cultures to produce high-value natural products is suggested to be a safe, low-cost, and environmentally friendly route to produce a wide range of chemicals. Given that the expression of heterologous biosynthetic pathways in plant tissue culture is limited by a lack of detailed protocols, the biosynthesis of high-value metabolites in plant cell culture is constrained compared with that in microbes. However, both Arabidopsis thaliana and Nicotiana benthamiana can be efficiently transformed with multigene constructs to produce high-value natural products in stable plant cell cultures. This chapter provides a detailed protocol as to how to engineer the plant cell culture as bio-factories for metabolite biosynthesis.

Evaluation of Salicylic Acid and Methyl Jasmonate as Elicitors in Phyllanthus acuminatus Hairy Roots by Non-Targeted Analysis Using High-Resolution Mass Spectrometry.

Phyllanthus acuminatus has been studied for its vast medical and industrial potential. Phytochemical investigations reveal that the genus is a rich source of lignans, flavonoids, phenolics, terpenoids, and other metabolites. However, the phytochemical profile elucidation of this species still needs further research. The use of eliciting compounds such as salicylic acid and methyl jasmonate has managed to increase the production of secondary metabolites in plant cell cultures. Hairy roots of Phyllanthus acuminatus were produced in 250 mL flasks with a 16 h light/8 h darkness photoperiod under diffused light with a culture time of four weeks. The elicitors salicylic acid and methyl jasmonate were tested in 50 μM and 200 μM concentrations. Non-targeted analysis was done for the different treatments using HR-MS. Identified metabolites were grouped in phenylpropanoids, phenols, and mucic acids, and statistical analysis of relative concentrations was achieved. A significant change in phenols' relative concentrations appeared in the elicitations with salicylic acid. Because of the elicitation treatment, specific compounds increased their concentrations, some of which have known pharmacological effects and are used in treating chronic diseases. The best elicitation treatment was salicylic acid 50 μM as it increased by more than 100% the general content of phenols and phenylpropanoid derivates and triplicates the concentration of mucic acid derivates in treated hairy root extracts. The application of non-targeted analysis showed interesting changes in phytochemical concentration due to elicitation in Phyllanthus acuminatus hairy roots.

Identification of gene markers to predict proanthocyanidins biosynthesis in cell culture of Croton lechleri

Croton lechleri Müll. Arg. is a traditional medicinal tree growing in South America, which produces a deep red sap known as Dragon’s Blood Sap (DBS) used for its many therapeutic benefits. DBS bioactivity is mainly due to the high content of proanthocyanidins (PAs), important plant secondary metabolites involved in plant defence, but also utilised as antioxidant for human health. Since the process to directly obtain DBS from the C. lechleri tree is expensive, time-consuming and strongly dependent on the environmental conditions, the use of plant cell cultures for DBS production seems to represent a suitable alternative. The aptitude of plant cell cultures to synthesize specific secondary metabolites can be elicited by different types of abiotic stresses. In this study, the C. lechleri main genes putatively involved in the PAs biosynthetic pathway were identified and their expression was evaluated in both calli in solid medium and cell culture suspensions to identify the most suitable predictive gene markers for PAs biosynthesis. C. lechleri cell cultures were grown and elicited by N-starvation to induce the production of PAs and were then monitored by assessing the expression of previously identified gene markers, by quantifying the content of PAs and measuring few phenotypical parameters. The results show a new perspective on how PA gene transcript abundance could be used to predict the biosynthesis of useful secondary metabolites in plant cell cultures and to speed up the screening of putative elicitors.

Summary of the Frontier Introduction of Preparation of Secondary Metabolites in Plant Cell Culture

Plant cell culture technology is a technology that applies the research results of cell engineering to produce plant biological products at the cellular level. In recent years, the secondary metabolites of plants have attracted more and more attention. The use of plant cell culture technology is a fast and efficient method of producing secondary metabolites.

Mathematical Modeling of Growth and Paclitaxel Biosynthesis in Corylus avellana Cell Culture Responding to Fungal Elicitors Using Multilayer Perceptron-Genetic Algorithm.

Paclitaxel is the top-selling anticancer medicine in the world. In vitro culture of Corylus avellana has been made known as a promising and inexpensive strategy for producing paclitaxel. Fungal elicitors have been named as the most efficient strategy for enhancing the biosynthesis of secondary metabolites in plant cell culture. In this study, endophytic fungal strain HEF17 was isolated from C. avellana and identified as Camarosporomyces flavigenus. C. avellana cell suspension culture (CSC) elicited with cell extract (CE) and culture filtrate (CF) derived from strain HEF17, either individually or combined treatment, in mid and late log phase was processed for modeling and optimizing growth and paclitaxel biosynthesis regarding CE and CF concentration levels, elicitor adding day, and CSC harvesting time using multilayer perceptron-genetic algorithm (MLP-GA). The results displayed higher accuracy of MLP-GA models (0.89–0.95) than regression models (0.56–0.85). The great accordance between the predicted and observed values of output variables (dry weight, intracellular, extracellular and total yield of paclitaxel, and also extracellular paclitaxel portion) for both training and testing subsets supported the excellent performance of developed MLP-GA models. MLP-GA method presented a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis. An Excel® estimator, HCC-paclitaxel, was designed based on MLP-GA model as an easy-to-use tool for predicting paclitaxel biosynthesis in C. avellana CSC responding to fungal elicitors.

Effects of methyl jasmonate and salicylic acid on the production of camphor and phenolic compounds in cell suspension culture of endemic Turkish yarrow(Achilleagypsicola) species

Elicitors have been widely used as biotic and abiotic stimulants in triggering the production of secondary metabolites in plant cell culture. The present study aimed to enhance the production of camphor and phenolic compounds and cell growth using methyl jasmonate (MeJA) and salicylic acid (SA) in the cell suspension culture of Turkish endemic species Achillea gypsicola. Various concentrations (0, 10, 50, and 100 μM) of MeJA and SA were applied to 8-day-old cell cultures. The camphor and phenolic compound contents were determined using a headspace gas chromatographic-mass spectrometer device and spectrophotometer. Increasing doses of MeJA and SA significantly enhanced the accumulation of the camphor and phenolic compounds in general. The highest amount of camphor accumulation occurred in cells treated with 100 μM MeJA (0.3449 μg/g) and 50 μM SA (0.3816 μg/g). Increasing concentrations of MeJA resulted in a significant decrease in the total anthocyanin when compared to the initial culture. The present study showed that MeJA and SA could effectively be used as potent elicitors to enhance the production of camphor and phenolic compounds, along with cell growth, in cell suspension cultures of the endemic Turkish yarrow species Achillea gypsicola.

Elicitors derived from endophytic fungi Chaetomium globosum and Paraconiothyrium brasiliense enhance paclitaxel production in Corylus avellana cell suspension culture

Paclitaxel is an impressive chemotherapeutic agent that shows biological activity against a wide range of cancers. Cell suspension culture of Corylus avellana has been reported as an alternative source for production of the valuable secondary metabolite, paclitaxel. The fungal elicitors have been considered as the most impressive strategy for enhancement of secondary metabolites in plant cell culture. In this study, two endophytic fungal strains YEF20 and HEF114 were isolated from Taxus baccata and C. avellana, respectively. The isolates YEF20 and HEF114 were identified as Chaetomium globosum and Paraconiothyrium brasiliense, respectively by sequencing of ITS1-5.8S-ITS2 rDNA region. This is the first report of P. brasiliense on C. avellana tree. Also, this study presents the positive effect of fungal elicitors on paclitaxel production in C. avellana for the first time. The effect of fungal elicitors on paclitaxel production was dependent on fungal species, and also type, concentration and adding time of elicitors to cell culture. The highest total yield of paclitaxel in cell culture treated with cell extract (CE) of C. globosum (291.5 µg L−1) was obtained by using 10% (v/v) of this fungal elicitor on 17th day of cell culture cycle (late log-phase), which was about 4.1 times obtained in the control culture. The cell culture treated with C. globosum CE had an average growth rate of 0.491 g L−1 day−1, i.e. 12.3% lower than that in the control. Among the fungal elicitors, 10% (v/v) CE of C. globosum on 17th day of culture cycle showed the best results with respect to extracellular paclitaxel portion (44.0%). Paclitaxel secretion to culture medium is essential for large-scale steady production of paclitaxel.

Enhancing taxane biosynthesis in cell suspension culture of Taxus chinensis by overexpressing the neutral/alkaline invertase gene

Sucrose metabolism generates material and energy foundations and regulates the biosynthesis of secondary metabolites in plant cell cultures. However, this regulation mechanism is unclear. The neutral/alkaline invertase (NINV) gene is among the key enzymes in sucrose hydrolysis. Thus, the complete open reading frame (ORF) of this gene was obtained from Taxus chinensis cells. This gene was also successfully expressed in Escherichia coli. The expressed protein was verified through the function of sucrose cleavage. Furthermore, the transcriptional levels of the NINV gene can respond to the sucrose concentration changes and relate to taxane biosynthesis in T. chinensis cells. Subsequently, we developed the gene expression vector and introduced it into T. chinensis suspension cells to confirm the role of the NINV gene in taxane biosynthesis. Results indicated that this gene was overexpressed and that it significantly enhanced the expression of the taxadiene synthase (TAS) gene, as well as the biosynthesis of the seven detected individual taxanes. Thus, NINV-mediated sucrose metabolism can promote the biosynthesis of secondary metabolites. This study presents new concepts and approaches to increase the production of secondary metabolites in plant cell cultures.

Application of cell technologies for production of plant-derived bioactive substances of plant origin

Bioactive substances (BAS) of plant origin are known to play a very important role in modern medicine. Their use, however, is often limited by availability of plant resources and may jeopardize rare species of medicinal plants. Plant cell cultures can serve as a renewable source of valuable secondary metabolites. To the date, however, only few examples of their commercial use are known. The main reasons for such a situation are the insufficient production of secondary metabolites and high cultivation costs. It is possible to increase the performance of plant cell cultures by one or two orders of magnitude using traditional methods, such as selection of highly productive strains, optimization of the medium composition, elicitation, and addition of precursors of secondary metabolite biosynthesis. The progress in molecular biology methods brought about the advent of new means for increasing of the productivity of cell cultures based on the methods of metabolic engineering. Thus, overexpression of genes encoding the enzymes involved in the synthesis of the target product or, by contrast, repression of these genes significantly influences the cell biosynthetic capacity in vitro. Nevertheless, the attempts of the production of many secondary metabolites in plant cell culture were unsuccessful so far, probably due to the peculiarities of the cell culture as an artificial population of plant somatic cells. The use of plant organ culture or transformed roots (hairy root) could turn to be a considerably more efficient solution for this problem. The production of plant-derived secondary metabolites in yeast or bacteria transformed with plant genes is being studied currently. Although the attempts to use metabolic engineering methods were not particularly successful so far, new insights in biochemistry and physiology of secondary metabolism, particularly in regulation and compartmentation of secondary metabolite synthesis as well as mechanisms of their transport and storage make these approaches promising.

Stimulation of anthocyanin synthesis in grape (Vitis vinifera) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.

Effects of Pulsed Electric Field on Secondary Metabolism of Vitis vinifera L. cv. Gamay Fréaux Suspension Culture and Exudates

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.

POSSIBILITIES OF PRACTICAL USE OF JASMONATES IN PRODUCTION OF FRUITS AND VEGETABLES

Jasmonates, mostly jasmonic acid (JA) and methyl jasmonate (JA-Me), are important signal transducers and greatly stimulate the biosynthesis of a wide range of secondary metabolites in plant cell cultures and in different intact plants. In this review we focused on the role of jasmonates in regulation of some physiological processes in growth and development of fruits and vegetables. For example, preharvest application of JA-Me improved fruit quality of blackberry and strawberry by stimulation of the biosynthesis of flavonoids and anthocyanins, and increased antioxidant capacities. In the case of sweet basil, JA-Me enhanced accumulation of alkaloids, terpenoids and phenolics. Moreover, JA-Me has been demonstrated to increase anthocyanin content in apple and strawberry fruits. It is well known that flavonoids, mostly anthocyanins, show preventive and therapeutic activities in many illness, for example extracts of blackberry fruits showed anticancer activity against human lung cells and leukemia cells. Besides, it has been found that exogenous JA-Me inhibits lycopene in tomatoes and stimulates β-carotene accumulation in tomatoes, apples, banana, guava, mango, and greatly stimulates ethylene production in tomatoes at different stages of ripening, as well as in preclimacteric apples, Japanese plum, and mangoes. JA-Me appears to be effective in reducing chilling injury in avocados, grapefruits, mangoes, peppers, cucumbers and others. JA-Me inhibits postharvest sprouting and improves storage quality of radishes. It is interesting also that JA-Me extends shelf life and reduces microbial contamination of fresh-cut celery, peppers, pineapple and tomatoes. Cooperative cross-talk among jasmonates and other phytohormones, especially ethylene occurs in regulation of growth and development and in defence responses against a wide of abiotic and biotic stressors. Moreover, it should be mentioned that jasmonates have medical importance due to inducing suppression of proliferation and death in several human cancer cells (leukemia, prostate, breast, lung and melanoma cell lines). These recently published findings suggest that the natural jasmonates may potentially be a novel class of anticancer drugs.

Uptake of nicotine from suspension culture ofNicotiana tabacumby molecularly imprinted polymers

The aim was to use molecularly imprinted polymers (MIPs) for the selective recovery of nicotine in plant cell cultures. MIPs can selectively uptake nicotine from suspension cultures of N. tabacum, and therefore may be useful for improving levels of secondary metabolites in plant cell cultures. Suspension cultures of N. tabacum were initiated from callus and maintained in liquid Murashige and Skoog (MS) media containing 3% w/v sucrose, 0.1 mg/l alpha-naphthaleneacetic acid acid (NAA) and 0.25 mg/l kinetin. Tween 80 at 1% was used for permeabilisation of cell cultures. Pre-weighed XAD-2 and two types of synthesized polymers, MIPs (A and B with one and two functional monomers, respectively) and corresponding non-imprinted polymers (NIPs), A and B, were introduced aseptically into the permeabilised suspension cultures of N. tabacum, the nicotine contents of polymers were determined by gas chromatography and the adsorption yield of polymers were determined. Cell cultures of N. tabacum accumulated nicotine alkaloid intracellularly in varying levels, 6.8-14.9 mg/l fresh weight. MIPs were able to uptake 50-70% of released nicotine in suspension cultures of N. tabacum, whereas XAD-2 recovered only 30-40%. The total levels of accumulated nicotine were enhanced up to 20 mg/l by simultaneous use of Tween 80 and MIPs. The findings indicate the potential use of MIPs to uptake nicotine from suspension cultures of N. tabacum, and increase productivity of secondary metabolites in plant cell cultures.

Enantiomeric separation of racemic 1-benzyl- N-methyltetrahydroisoquinolines on chiral columns and chiral purity determinations of the O-methylated metabolites in plant cell cultures by HPLC-CD on-line coupling in combination with HPLC-MS

Effective enantiomeric separations of 1-benzyl- N-methyltetrahydroisoquinolines were achieved using commercially available Chiralcel OD-H and OJ-H columns. Online LC-CD analysis allowed for the establishment of a correlation between the absolute configuration of the separated enantiomers and their characteristic CD transitions. LC-MS combined with LC-CD analysis permitted chiral purity determinations of O-methylated metabolites of nine phenolic 1-benzyl- N-methyltetrahydroisoquinolines in cell cultures of Corydalis, Macleaya, and Nandina species.

Agrobacterium rhizogenesMediated Genetic transformation ofAbrus precatoriusL

Abrus precatorius L. known as Indian liquorice is a common deciduous vine containing sweet principle compound known as glycyrrhizin. Hence it can be used as a very good substitute for Liquorice. Genetic transformation has proved to be an effective way to enhance secondary metabolites in plant cell cultures. The transformation of Abrus precatorius L. mediated by Agrobacterium rhizogenes was studied using three bacterial strains i.e. MTCC 532, MTCCC 2364 and NCIM 5140. Putative hairy roots were obtained after the transformation. The effects of bacterial strains, bacterial concentration, acetosyringone and co-cultivation pH on transformation of Abrus were investigated. Co-cultivation with Strain MTCC 532 for 2 days with 100 µmol L -1 acetosyringone at pH 6.5 provided the optimal conditions under which transformation frequency approached 84%.

Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24 h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7 ± 42.0 mg/L. Also, ( S)-methylcoclaurine-3′-hydroxylase (CYP80B1) and 3′-hydroxy-(S)- N-methylcoclaurine-4′- O-methyltransferase (4′OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.

Phycocyanin, a new elicitor for capsaicin and anthocyanin accumulation in plant cell cultures

Elicitors of both fungal and bacterial origin that is, polysaccharides, proteins and fatty acids, are widely used for enhancement of secondary metabolites in plant cell cultures. In the present study, phycocyanin - a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis - was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in Capsicum frutescens and Daucus carota cell cultures respectively. Phycocyanin at 0.3, 0.6 and 1.2 mg% in capsicum cell cultures elicited a more than two-fold increase in capsaicin content with maximum productivity of 192 µg/g fresh weight. Similarly in Daucus carota cell cultures a two-fold increase in anthocyanin content was obtained at 0.3 mg% with a maximum productivity of 24.8 mg% on a dry-weight basis. In both the systems, phycocyanin showed an early elicitation of secondary metabolites.

Characterization of abscisic acid and metabolites by combined liquid chromatography-mass spectrometry with ion-spray and plasma-spray ionization techniques

Liquid chromatographic-mass spectrometric methods have been developed for the characterization of abscisic acid (ABA) and metabolites in plant cell cultures using plasma-spray and ion-spray ionization techniques. Both techniques provide useful mass spectral data for ABA and its acidic metabolites. The most intense ions in these spectra are represented by [M + H] + and [M + H − H 2O] +. The limit of detection for ABA, using plasma-spray and ion-spray was 5 and 9 ng, respectively, for full scans and 0.5 and 1 ng, respectively, for selected-ion recording of m/z 265, the protonated molecular ion. Ion-spray also provided intense protonated molecular ions for the neutral ABA metabolites whereas plasma-spray did not. Spectra acquired using both techniques showed intense ions indicating the presence of the ABA moiety in these neutral conjugates.

Use of Nurse Cultures to Select for Papaver somniferum Cell lines Capable of Enhanced Sanguinarine Production

Summary A nurse culture technique was used in which cell lines of Papaver somniferum capable of producing 12 % sanguinarine (dry weight basis) have been isolated. Cell aggregates composed of 4 to 16 cells were removed from suspension cultures and inoculated on a sterile cell support located on top of the actively growing callus. Approximately 25 % of the cultured aggregates developed into callus cultures, and nearly all of these were resuspended successfully. Up to a 30-fold range in the sanguinarine yields from these cell lines was observed, indicating that considerable cell variation was present in the cells of the original parental line. Evidence of aneuploidy was observed, which may explain this cellular variation. This report provides proof that aggregate selection is a valuable tool in increasing the yields of secondary metabolites in plant cell cultures.

Aspects Affecting Biosynthesis and Biotransformation of Secondary Metabolites in Plant Cell Cultures

(1984). Aspects Affecting Biosynthesis and Biotransformation of Secondary Metabolites in Plant Cell Cultures. Critical Reviews in Biotechnology: Vol. 2, No. 2, pp. 105-118.