Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Temporal lag between gene expression and metabolite accumulation in flavonol biosynthesis of Arabidopsis roots

The temporal lag between gene expression and metabolite accumulation has been estimated in flavonol biosynthesis, but the time difference between these events is unclear. In the present study, we investigated the expression of flavonol biosynthetic genes ELONGATED HYPOCOTYL5, MYELOBLASTOSIS PROYEIN12/PRODUCTION OF FLAVONOL GLYCOSYDES1, CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE, and FLAVONOL SYNTHASE1, and the accumulation of flavonol glycosides (kaempferol and quercetin glycosides) in time-series samples of Arabidopsis thaliana roots. All genes started to be expressed within 3 h after sequential light irradiation (HAS) and reached their maximum expression levels at 12 HAS, and the accumulation of the flavonol glycosides started at 6 HAS. Metabolome analysis using liquid chromatography-mass spectrometry showed that the accumulation of kaempferol 3-O-glucoside-7-O-rhamnoside and kaempferol 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside reached their maximum levels at 48 HAS, whereas other flavonol glycosides, such as kaempferol/quercetin 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3-O-rhamnosyl (1 → 2) glucoside-7-O-rhamnoside, increased gradually until 96 HAS. These results show that the expression of the flavonol genes is an early response against light exposure, and that the accumulation of the flavonol glycosides is a late response.     

Cytochalasin D enhances the accumulation of a protease‐resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway

The conversion of a host‐encoded PrPsen (protease‐sensitive cellular prion protein) into a PrPres (protease‐resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion‐infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule‐disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3‐kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D‐induced enhancement of PrPres accumulation. Cytochalasin D‐induced extension of neurite‐like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion‐infected cells.     

Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad

Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 μM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 μM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets.     

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis

BACKGROUND: Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). METHODS: To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. RESULTS: Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. CONCLUSIONS: Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.     

Sweating treatment enhances citrus fruit disease resistance by inducing the accumulation of amino acids and salicylic acid‐induced resistance pathway

To clarify the mechanism of fruit disease resistance activated by sweating treatment, ‘Guoqing NO.1’ Satsuma mandarin (Citrus unshiu Marc.) fruits were treated by sweating, which is a traditional prestorage treatment in China. Subsequently, we performed inoculation and physiological characterization, two‐dimensional gel electrophoresis (2‐DE) proteomics analysis and metabonomics analysis based on gas chromatography coupled to mass spectrometry (GC‐MS) and high‐performance liquid chromatography/electrospray ionization‐time of flight‐mass spectrometry (HPLC‐qTOF‐MS). The results showed that sweating treatment significantly inhibited pathogen infection without negatively affecting the fruit commercial quality. In addition, sweating treatment rapidly promoted the accumulation of amino acids (such as proline and serine). Meanwhile, hydrogen peroxide (H₂O₂) and salicylic acid (SA) were significantly accumulated in the sweating‐treated fruit. Thereafter, some stress‐response proteins and metabolites [such as ascorbate peroxidase (APX), β‐1,3‐glucanase, vanillic acid and rutin] which can be induced by SA were also significantly increased in the sweating‐treated fruit. Taken together, the disease resistance induced by sweating treatment might be attributed to: (1) the induction of the accumulation of amino acids; and (2) the accumulation of SA and subsequent activation of SA‐induced resistance pathway, which can induce the stress‐response proteins and metabolites that can directly inhibit pathogen development.