Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro.

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 µmol m-2 s-1 by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 µmol m-2 s-1 than at 65 µmol m-2 s-1, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 µmol m-2 s-1 stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata. Graphical abstract.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

Assessment of the phenolic content, mutagenicity and genotoxicity of ethanolic extracts of stem bark and leaves from Strychnos pseudoquina A. St.-hil.

Strychnos pseudoquina is a plant species whose stem bark is used as bitter tonic beverage. The phytochemical analysis, as well as quantification of phenolic constituents and antioxidant activity of ethanolic extracts from S. pseudoquina stem bark, and leaves were conducted. The extracts were tested for mutagenicity (Ames test) and DNA-damaging activity (Plasmid Cleavage test). Leaves recorded the largest amount of flavonoids. The performed high-performance liquid chromatography (HPLC) showed flavonoids such as isorhamnetin and strychnobiflavone (phytochemical markers of the investigated species) in stem barks, but not in leaves. The proanthocyanidin content and antioxidant activity were significantly higher in stem barks than in leaves. Stem bark and leaf extracts presented mutagenic activity against TA98 and TA100 strains with, and without, metabolic activation (S9). The Plasmid Cleavage test did not indicate DNA-damaging activity. Our results suggest that extracts deriving from S. pseudoquina should be used with extreme caution, mainly the stem bark extract, which is widely used in folk medicine.