Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

Abstract

Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50-100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a "natural" elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production.

Keywords: abiotic elicitors; bioactive metabolites; cellular agriculture; cosmeceutics; plant cell extracts.

Figure 1

(A–D) Appearance of cardoon calli on solid Gamborg B5 culture medium under (A) chilling stress at 6 °C for 7 and 14 days or (B) saline stress of 0, 50, 100 or 150 mM NaCl for 14 and 28 days. Growth changes of cardoon calli subcultured on solid Gamborg B5 culture medium under (C) chilling stress at 6 °C for 7 and 14 days or (D) saline stress of 0, 50, 100 or 150 mM NaCl for 14 and 28 days. Each value represents the mean of nine biological replicates ± SD/10. Statistical significance between samples within each time point was determined by one-way ANOVA with Tukey’s post hoc test. Different letters indicate significant differences (p < 0.05).

Figure 2

(A,B) Relative expression changes of CcHQT and CcF3′H biosynthetic genes in cardoon callus cultures in response to (A) chilling stress for 7 and 14 days at 6 °C or (B) saline stress at 0, 50, 100 or 150 mM NaCl for 14 and 28 days. Total RNA was extracted from unstressed (CTR) and treated calli (chilling or salt stress) and unstressed calli at each time point were used as controls. Each value represents the mean of three biological replicates ± SD. Statistical significance for each gene between treatments within each time point was determined by using one-way ANOVA with Tukey’s post hoc test. Different letters indicate significant difference (p-value < 0.05).

Figure 3

(A,B) Changes in antioxidant power measured via 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays in cardoon callus cultures in response to (A) 7–14 days chilling at 6 °C or (B) 14–28 days of 0, 50, 100, or 150 mM NaCl stress. Values are expressed as TEAC (Trolox^®-equivalent antioxidant capacity, mmol Trolox Kg⁻¹ DW). Each value represents the mean of three biological and two technical replicates ± SD. Statistical significance for each activity between treatments within each time point was determined by using one-way ANOVA with Tukey’s post hoc test. Different letters indicate significant difference (p-value < 0.05).

Figure 4

(A,B) Non-clustered heat map of major changes exerted by elicitation by abiotic stresses in cardoon calli. Heat maps for (A) 6 °C chilling stress treatment for 7 or 14 days, and (B) 0, 50, 100, or 150 mM NaCl salt stress treatment for 14 or 28 days. The columns of the heat maps represent control and stress treatments at the different time points, the rows represent different physiological and metabolic parameters (growth, lipid peroxidation MDA, total polyphenols, DPPH, ABTS, and FRAP assay). Each cell is colored based on the value in that sample. The intensity of color is representative of degree of differences; the value of 0 denotes no difference, with higher values corresponding to larger differences. Values from lower to higher are indicated by light green to blue for chilling stress and yellow to red for salt stress.

Figure 5

Protection activity of cardoon calli extracts on cell viability. Human keratinocytes were treated with the test compounds at different concentrations. The cells were processed as described in the Material and Methods section to measure cell viability (MTT assay). Each value represents the mean of three biological replicates ± SD. CTR, control unstressed cells; 0 mM and 100 mM NaCl, saline stress treatments.

Figure 6

Effects of cardoon calli extracts on increasing the production of (A) pro-collagen I compared to TGFβ treatment and (B) AQP3 protein in comparison with Rubus idaeus as positive control; the quantization was performed by detection with antibody labeled with fluorophore. The bars represent the mean of three biological replicates ± SD. Statistical significance was determined between treatments within each time point by using one-way ANOVA with Tukey’s post hoc test. Different letters indicate significant difference (p-value < 0.05). CTR, control unstressed cells; 0 mM and 100 mM NaCl, saline stress treatment.