Morphophysiology and 20-hydroxyecdysone production in Pfaffia glomerata (Spreng.) Pedersen in response to CO2-enrichment and drought stress
Authors:   , Silva Tatiane Dulcineia 
Published: September 2022

Anthropogenic activities have been catalyzing climate changes, owing to increased atmospheric carbon dioxide concentration ([CO 2 ]). In respect to CO 2 , its general effects on the regulation of plant growth, development and metabolism are widely explored, especially in agricultural crops. However, the changes in medicinal species under elevated [CO 2 ] are still poorly understood. Pfaffia glomerata (Spreng.) Pedersen, a plant species native to Brazil and popularly known as Brazilian ginseng, stands out by the production of 20-hydroxyecdysone (20E), a phytoecdysteroid molecule with proven therapeutic and nutraceutical activities in mammals. Previous findings from our research group revealed that in vitro CO 2 -enrichment led to physiological changes in P. glomerata. We hypothesize that CO 2 -enrichment affects the production of primary and secondary metabolites, such as 20E, in P. glomerata. Therefore, the objective here was to evaluate the effect of the CO 2 -enriched atmosphere on morphophysiological, biochemical, structural, molecular aspects, as well as in water deficit tolerance responses. For this, two independent experiments were conducted in open top chambers (OTCs). For both experiments, clonally micropropagation-derived plantlets from the in vitro germplasm bank were used. In experiment I, we assessed the influence of two CO 2 concentrations: ambient (a[CO 2 ], ± 400 µmol mol -1 CO 2 ) and elevated (e[CO 2 ] ± 800 µmol mol -1 CO 2 ). After approximately 21 days of cultivation in OTC, our data showed that e[CO 2 ] increased photosynthesis (A N ), and water use efficiency (WUE), biomass accumulation, photosynthetic pigments, carbohydrate content, expression of genes of the lignin biosynthetic pathway, and induced changes in the proteomic profile. On the other hand, e[CO 2 ] promoted reduction in total protein, amino acids and 20E contents. In experiment II, the physiological and biochemical responses of plants exposed to combined e[CO 2 ] and under water deficit were analyzed. During the first 14 days of cultivation, all plants were regularly irrigated to maintain soil water at field capacity. Then, the plants were subjected for 21 days to the following treatments: (I) a[CO 2 ] and well-watered (a[CO 2 ]WW); (II) e[CO 2 ] and well- watered (e[CO 2 ]WW); (III) a[CO 2 ] and drought stressed (a[CO 2 ]D); and (IV) e[CO 2 ] and drought stressed (e[CO 2 ]D). Our data demonstrate that e[CO 2 ] mitigates drought impacts by reducing stomatal conductance (g S ), improving WUE, and promoting less negative water potentials. Although the reduction in gs decreased water loss by evapotranspiration, it negatively affected A N , and consequently plant growth. Hyperspectral analysis corroborates these findings and is a great non-destructive tool to analyse the effects of drought on plants. We found that P. glomerata exhibits different drought response mechanisms depending on [CO 2 ]. Plants exposed to a[CO 2 ]D increased root/shoot ratio, stem rustification, non- enzymatic antioxidant system by increased anthocyanin content and proteins of ascorbate and glutathione metabolism. Conversely, plants under e[CO 2 ]D invested strongly in osmoregulatory metabolites (e.g. soluble sugars and amino acids). We found that e[CO 2 ] associated to drought promoted changes in the proteomic profile, dramatically affecting the accumulation of stress response proteins. On the other hand, we show that drought positively affected the activity of antioxidant enzymes (CAT and SOD) and 20E content in both [CO 2 ] in P. glomerata plants. However, only in plants exposed to e[CO 2 ]D was there an increase in 20E production, overcoming the biomass limitation caused by this stress. Here, we report for the first time, the up-regulation of cytochrome P450 CYP72A219-like protein in plants grown in the combination e[CO 2 ]D. Therefore, we hypothesize a relationship of this protein with 20E biosynthesis and hypothesize possible ROS signaling (indirectly by increased CAT and SOD) under abiotic stress condition. These data provide relevant information in elucidating the pathway of 20E biosynthesis, as well as enabling biotechnological strategies to increase the production of this metabolite in P. glomerata plants. Keywords: Brazilian ginseng. 20-hydroxyecdysone. Phytoecdysteroids. Water stress. CO 2 enrichment. Secondary metabolism.