On the mechanism of the cell cycle control of suspension-cultured tobacco cells after exposure to static magnetic field.

One of the main sites of the magnetic fields influence on living cells is the cell cycle. The intensity of this influence however, varies depending on the cell type and the duration of the treatment. Suspension of cultured tobacco cells (Nicotiana tabacum cv. Barley 21) were synchronized via sucrose starvation at their stationary growth phase. The cells were then exposed to 0.2 m T SMF up to 24 h. The progression of different cell cycle phases was monitored through flow cytometry in a time course manner. Expression of cell cycle controlling genes and amounts of certain signaling molecules were measured as well. Exposure to SMF delayed G1.S transition which was accompanied by decrease of cyclin-dependent kinases A (CDK A) and D-type cyclin, but an increase in the adenylyl cyclase (AC), transcription factor E2F, retinoblastoma protein (Rbp), and CDK-inhibitor protein 21 (p21) transcript accumulation. Exposure to SMF also increased the contents of nitric oxide (NO), hydrogen peroxide (H2O2), and salicylic acid (SA), compared to the control group. The results suggest a signaling pathway triggered by SMF starting from accumulation of NO and H2O2 followed by downstream events including the increase of cyclic nucleotides and subsequent decrease of both CDKA and CycD.

Piriformospora indica cell wall modulates gene expression and metabolite profile in Linum album hairy roots.

MAIN CONCLUSION: Elicitation of Linum album hairy roots by Piriformospora indica cell wall induced the target genes and specific metabolites in phenylpropanoid pathway and shifted the amino acid metabolism toward the phenolic compound production. Plants have evolved complex mechanisms to defend themselves against various biotic stresses. One of these responses is the production of metabolites that act as defense compounds. Manipulation of plant cell cultures by biotic elicitors is a useful strategy for improving the production of valuable secondary metabolites. This study focused on hairy root culture of Linum album, an important source for lignans. The effects of cell wall elicitor extracted from Piriformospora indica on phenylpropanoid derivatives were evaluated to identify metabolic traits related to biotic stress tolerance. Significant increases in lignin, lignans; lariciresinol, podophyllotoxin, and 6-methoxy podophyllotoxin; phenolic acids: cinnamic acid, ferulic acid, and salicylic acid; flavonoids: myricetin, kaempferol, and diosmin were observed in response to the fungal elicitor. In addition, the gene expression levels of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, and pinoresinol-lariciresinol reductase significantly increased after elicitation. The composition of free amino acids was altered under the elicitation. Phenylalanine and tyrosine, as precursors of phenylpropanoid metabolites, were increased, but alanine, serine, and glutamic acid significantly decreased in response to the fungal elicitor, suggesting that the amino acid pathway may be shifted toward biosynthesis of aromatic amino acids and precursors of the phenylpropanoid pathway. These results provided evidence that up-regulation of genes involved in the phenylpropanoid pathway in response to the fungal elicitor resulted in enhanced metabolic responses associated with the protection in L. album. This approach can also be applied to improve lignan production.

A comparative study of biotechnological approaches for producing valuable flavonoids in Prosopis farcta.

The callus and hairy root cultures of Prosopis farcta were established to develop effective strategies to enhance its valuable and medicinally important flavonoid compounds. For callus induction, the hypocotyl, cotyledon and shoot explants were subjected to different plant hormones, naphthalene acetic acid (NAA), benzylaminopurine (BAP), kinetin and dichlorophenoxyacetic acid (2,4-D). Greater callus induction was obtained from hypocotyl explants on MS medium containing 3.0 mg L-1 NAA + 2.0 mg L-1 BAP. With the addition of 0.5 mg L-1 asparagine to this medium, the maximum callus growth was achieved. Hairy root culture of P. farcta was performed using transformation of different explants with strains of Agrobacterium rhizogenes LBA9404, A4, AR15834. The AR15834 strain was more effective for hairy root induction where it caused hairy root formation on 59% of the infected cotyledon explants. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and callus cultures of P. farcta. The colorimetric analysis showed that the content of total flavonoids of hairy roots was 1.54 and 2.52 times higher than in seedling roots and callus, respectively. The presence of flavonoids was verified by LC/MS in positive ion mode. The results showed that flavonoid composition was different in the roots and callus. Naringenin was the major constituent in callus, whereas resveratrol, quercetin and myricetin were the most abundant compounds found in hairy roots. The main objective of this research was to establish hairy roots in P. farcta to synthesize flavonoids at levels comparable to in vitro-grown roots. The present study also opens up a way to further improve the production of pharmaceutically valuable flavonoids and to produce desired metabolites using the hairy root culture system.

The contribution of cell wall composition in the expansion of Camellia sinensis seedlings roots in response to aluminum.

MAIN CONCLUSION: Treatment with aluminum triggers a unique response in tea seedlings resulting in biochemical modification of the cell wall, regulation of the activity of the loosening agents, and elongation of root. Unlike most terrestrial plants, tea (Camellia sinensis L.) responds to aluminum (Al) through the promotion of its root elongation; but the real mechanism(s) behind this phenomenon is not well understood. A plausible relationship between the modifications of the cell wall and the promotion of root elongation was examined in tea seedlings treated for 8 days with 400 µM Al. The mechanical properties of the cell wall, the composition of its polysaccharides and their capacity to absorb Al, the expression of genes, and the activities of the wall-modifying proteins were studied. With 6 h of the treatment, about 40% of the absorbed Al was bound to the cell wall; however, the amount did not increase thereafter. Meanwhile, the activity of pectin methylesterase, the level of pectin demethylation, the amounts and the average molecular mass of xyloglucan in the root apices significantly decreased upon exposure to Al, resulting in the reduction of Al binding sites. On the other hand, the activity and the gene expression of peroxidase decreased, whereas the activity and gene expression of xyloglucan-degrading enzymes, the expression of expansin A and the H +-ATPase4 genes increased in the Al-treated plants. Interestingly, it was accompanied by the increase of elastic and viscous extensibility of the root apices. From the results, it can be suggested that the biochemical modification of the cell walls reduces sites of Al binding to roots and triggers the activity of the loosening agents, thereby increasing the length of tea roots.

Favouring NO over H2O2 production will increase Pb tolerance in Prosopis farcta via altered primary metabolism.

Reactive oxygen species (ROS) and nitric oxide (NO) are known in triggering defense functions to detoxify heavy metal stresses. To investigate the relevance of ROS production, Pb treatment (400µM) alone and in combination with 400µM sodium ascorbate (Asc: as H2O2 scavenger) were given to hydroponically grown Prosopis farcta seedlings over a time course of 72h. Data presented here indicate that, the low extent of H2O2 due to scavenging by ascorbate, together with high level of NO improved Pb+Asc- treated Prosopis growth. Following the evoked potential of both the signals, significant increases in phenolic acids; caffeic, ferulic and salicylic acid were observed with Pb treatment; which are consistent with observed increase in lignin content and consequently with growth inhibition. In contrast, Pb+Asc treatment induced more flavonoids (quercetin, kaempferol, luteolin), diminished phenolic acids contents and also lignin. Elicited expression rate of phenylalanine ammonia-lyase gene (PAL) and also its enzymatic activity verified the induced phenylpropanoid metabolism by Pb and Pb+Asc treatments. In comparison with Pb stress, Asc+Pb application induced the high expression of arginine decarboxylase gene (ADC), in polyamines biosynthesis pathway, and conducted the N flow towards polyamines and γ-amino butyric acid (GABA). Examining the impact on enzyme activities, catalase, and guaiacol peroxidase; Pb+Asc reduced activity but this increased ascorbate peroxidase, and aconitase activity. Our observations are consistent with conditions favouring NO production and reduced H2O2 can improve Pb tolerance via wide-ranging effects on a primary metabolic network.

Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress.

Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects.

Optimization of podophyllotoxin extraction method from Linum album cell cultures.

CONTEXT: Suspension cultures of Linum album Kotschy ex Boiss. (Linaceae) accumulate podophyllotoxin (an anticancer agent) and could therefore serve as an alternative source of this important aryltetralin lactone lignan. OBJECTIVE: The present work compared four podophyllotoxin extraction methods and optimization of the best one by using single factor experiments. MATERIALS AND METHODS: Linum album cell cultures were established from in vitro plantlets and subcultured in MS medium with hormones every 7-8 days. Four podophyllotoxin extraction methods were assayed and the best one was optimized by single factor experiments, studying the effect of methanol concentration, extraction time, and sonication time. RESULTS: Cell cultures accumulated enough podophyllotoxin to be analyzed by HPLC. The methanol/dichloromethane and buffer extraction methods were found to be the best. Methanol alone and hot ethanol were not effective for extracting podophyllotoxin. DISCUSSION AND CONCLUSION: The optimized method based on methanol/dichloromethane extraction combined with HPLC quantification was able to determine small amounts of podophyllotoxin in Linum album cell cultures, showing that this system could constitute a possible alternative source of podophyllotoxin to Podophyllum (Berberidaceae).

Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.

Plants are a potential source of a large number of valuable secondary metabolites. In vitro cultures are being considered as an alternative to agricultural processes for studying valuable secondary metabolites. In this way, nutritive factors are important parameters influencing the production of these compounds in plants. Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine, and the growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were investigated. As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis. A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834. The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content. The alkaloid concentrations obtained in the hairy roots were 3-20 times higher than that in the plants at 35 mM of KNO3. Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants.