Time-dependent behavior of phenylpropanoid pathway in response to methyl jasmonate in Scrophularia striata cell cultures.

KEY MESSAGE: MeJA triggers a time-dependent behavior of the phenylpropanoid compounds. Plant cells produce a large number of metabolites in response to environmental factors. The cellular responses to environmental changes are orchestrated by signaling molecules, such as methyl jasmonate (MeJA). To understand how the MeJA changes the behavior of amino acids, carbohydrates, and phenylpropanoid compounds such as phenolic acids, phenylethanoid-glycosides, and flavonoids in Scrophularia striata cells; we monitored the metabolic responses for different times of exposure. In this study, we performed a time course analysis of metabolites and enzymes in S. striata cells exposed to MeJA (100 µM) and evaluated the metabolic flux towards carbon-rich secondary metabolites production. Moreover, we calculated the biosynthetic energy cost for free amino acids. Our results indicated that MeJA accelerates the sucrose degradation and directs the metabolic fluxes towards a pool of flavonoids and phenylethanoid glycosides through a change in enzyme behavior in the entry point and center of the phenylpropanoid pathway. MeJA also decreased and then raised the amino acid biosynthesis cost in S. striata cells in a time-dependent manner, indicating the cells evolve to utilize amino acids more economically by reducing cell growth. Finally, we classified the marked changes in the metabolites level and enzyme activities into three groups including early-, late-, and oscillatory-response groups to MeJA and summarized our findings as a model depicting pathway interactions during MeJA elicitation. Determination of metabolic levels in response to MeJA suggests that the changes in metabolic responses are time-dependent.

Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch.

Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS) medium containing 3.0 mg·L(-1) 6-benzyladenine (BA) in a combination with 2 mg·L(-1) 2,4-dichlorophenoxy acetic acid (2,4-D). From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa), salicylic acid (SA), and sodium nitroprusside (SNP) on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

Profiling of acidic (amino and phenolic acids) and phenylpropanoids production in response to methyl jasmonate-induced oxidative stress in Scrophularia striata suspension cells.

MAIN CONCLUSION: A metabolic profiling including calculation of energy cost of amino acids biosynthesis in cultured cells of Scrophularia striata showed that methyl jasmonate-inducible oxidative stress elicited secondary metabolites formation derived from phenylalanine and tyrosine and increased energy cost for these amino acids biosynthesis. Understanding of the metabolic pathways in cell culture of Scrophularia striata, an aromatic plant species, facilitates means of production of pharmaceutical metabolites under oxidative stress. In this study, we evaluated the effects of MeJA on the S. striata metabolic pathway and the responses to oxidative stress. Exposure to methyl jasmonate (MeJA) affects plant growth, effectively induces production of reactive oxygen species (ROS) and inserts oxidative stress at the cellular level which results in alteration of primary metabolites and production of phenylepropanoid compounds. Cells treated with MeJA indicated increase in the activities of three antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPx) as well as intracellular H2O2 and MDA contents compared with mock-treated cells. High performance liquid chromatography (HPLC)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed S. striata cells, e.g., general phenylpropanoid pathway, phenylethanoid-glycosides, lignans, and increased energy cost of biosynthesis and accumulation of amino acids. Furthermore, principal component analysis (PCA)-derived score plots demonstrated that MeJA affects cellular metabolism in S. striata cells and significantly alters metabolite composition under MeJA-inducible oxidative stress. These observations suggest that MeJA-elicited cell suspension cultures of S. striata balanced the production of primary and secondary metabolites in coordination with ROS-scavenging system.

Hyperhydricity in shoot cultures of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels.

An effective procedure for obtaining healthy shoots from nodal segments of Scrophularia yoshimurae is described. Nodal segments cultured on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg L(-1) benzyladenine (BA) and 0.2 mg L(-1) alpha-naphthaleneacetic acid (NAA) induced multiple shoots in conical flasks that differed in the way they were closed and sealed. Hermitically sealed culture vessels resulted in high hyperhydricity/vitrification. High concentrations of ethylene and CO2 were found to accumulate in these vessels. The hyperhydricity of the shoot cultures could be decreased by progressively ventilating the vessels. Exchange of gases was achieved by removing the Parafilm sealing without affecting sterility. This reduced the hyperhydricity rate and gave a good recovery of vitrified shoots, but resulted in decreased proliferation and a dehydration of proliferating nodal segments and the culture medium. The best number of normal shoots was observed when the parafilm was removed for gaseous exchange after four weeks of culture incubation. The results show that hyperhydricity in shoot cultures of S. yoshimurae could be prevented by sufficient gas exchange during culture.