Influence of peanut hairy root cultivars on prenylated stilbenoid production and the response mechanism for combining the elicitors of chitosan, methyl jasmonate, and cyclodextrin.

MAIN CONCLUSION: Peanut cultivars are known to produce stilbene compounds. Transcriptional control plays a key role in the early stages of the stress response mechanism, involving both PR-proteins and stilbene compounds. In this study, the production of stilbenoid compounds, especially prenylated, was investigated in two cultivars of peanut hairy root lines, designated as K2-K599 and T9-K599 elicited with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT + MeJA + CD. The antioxidant activities and stilbenoid content of both K2-K599 and T9-K599 hairy root lines increased significantly during the elicitation period. The T9-K599 hairy root line expressed higher ABTS and FRAP antioxidant activities than the K2-K599 line while the latter exhibited greater total phenolic content than the former at all-time points. Additionally, the K2-K599 line exhibited more stilbene compounds, including trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 than the T9-K599 line, which showed statistically significant differences at all-time points. Gene expression of the enzyme involved in the stilbene biosynthesis pathway (PAL, RS, RS3) was observed, responding early to elicitor treatment and the metabolic production of a high level of stilbenoid compounds at a later stage. The antioxidant enzyme (CuZn-SOD, APX, GPX) and pathogenesis-related protein (PR; PR4A, PR5, PR10, chitinase) genes were strongly expressed after elicitor treatment at 24 h and decreased with an increasing elicitation time. Investigation of the response mechanism illustrates that the elicitor treatment can affect various plant responses, including plant cell wall structure and integrity, antioxidant system, PR-proteins, and secondary plant metabolites at different time points after facing external environmental stimuli.

Production and antimicrobial activity of trans-resveratrol, trans-arachidin-1 and trans-arachidin-3 from elicited peanut hairy root cultures in shake flasks compared with bioreactors.

Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and secondary metabolite production. Enhanced production of stilbene compounds such as trans-resveratrol, trans-arachidin-1 and trans-arachidin-3 was achieved using an elicitor treatment procedure. Two different hairy root inoculum densities were investigated and compared between shake flask and bioreactor cultures. The lowest growth index was observed using a 20 g/L inoculum size in the bioreactor, which differed significantly from bioreactor of 5 g/L. Increasing the hairy root inoculum size from 5 g/L to 20 g/L in both the shake flask and bioreactor significantly improve antioxidant activity, phenolic content and stilbene compound levels. The highest ABTS and FRAP antioxidant activity, and levels of total phenolic compounds, trans-arachidin-1 and trans-arachidin-3 in the crude extract were demonstrated in shake flask cultures with a 20 g/L inoculum after elicitation for 72 h. The minimum inhibitory concentrations (MICs) of the crude extract to inhibit growth of foodborne microbes, S. aureus, S. typhimurium and E. coli, were 187.5, 250 and 500 µg/mL, respectively. This was due to the ability of the crude extract to disrupt the cell membrane, as observed by scanning electron microscopy (SEM) showing ruptured pores on the S. aureus and S. typhimurium cell surfaces. Moreover, the E. coli cell division process could be inhibited by the crude extract, which promoted an increase in cell size. A DNA nicking assay indicated that a 50 µg/mL concentration of the crude extract caused plasmid DNA damage that might be due to a genotoxic effect of the pro-oxidant activity of the crude extract.

Effect of light and mechanical stress in combination with chemical elicitors on the production of stilbene compounds and defensive responses in peanut hairy root culture.

Plants encounter diverse stressors simultaneously with changing environmental factors. The combined effect of different types of stresses can have a wide range of effects on plants. The present study demonstrated that various stress factors such as the combination of chemical elicitors, namely paraquat (PQ), methyl jasmonate (MeJA) and methyl-ß-cyclodextrin (CD), light exposure versus darkness, and mechanical shearing stress affected the defence response in peanut hairy root culture. The antioxidant activities were dramatically increased at all time points after hairy roots were subjected to elicitation with PQ + MeJA + CD under root cutting in both light and dark conditions. The stilbene compounds were highly increased in the culture medium after elicitor treatment of uncut hairy roots under dark conditions. In contrast to the high stilbene contents detected in culture medium under dark conditions, the transcription of the stilbene biosynthesis genes PAL, RS and RS3 was enhanced by the effect of light in uncut hairy root tissues. The antioxidant enzyme genes APX, GPX and CuZn-SOD of uncut and cut hairy roots were more highly expressed in light conditions than in dark conditions. The pathogenesis-related protein (PR)-encoding genes chitinase, PR4A, PR5 and PR10 of uncut hairy roots were highly expressed in response to light conditions compared to dark conditions at all time points. Recent evidence of the production of antioxidant stilbene compounds and defence response genes has implicated plant protective functions through defence responses under different stress challenges. Plant responses might therefore be regulated by the coordination of different signal responses through dynamic pathways.

Methyl jasmonate and cyclodextrin-mediated defense mechanism and protective effect in response to paraquat-induced stress in peanut hairy root.

Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidative stress. The present work elucidated a schematic diagram of the proposed pathway of peanut hairy root tissue treated with different elicitors; paraquat (PQ), methyl jasmonate (MeJA), and cyclodextrin (CD). The different elicitation approaches could provoke intrinsic stress in plant cells and might activate a distinct response pathway, allowing plants to overcome the deleterious effects of oxidative stress. Among all strategies, hairy root culture pretreated with PQ followed by application of MeJA plus CD showed an extensive induction of antioxidant defense mechanisms. The expression of the antioxidant enzyme genes and stilbene-synthesized enzyme genes were up-regulated in accordance with the dramatic increase in the production of stilbene compounds. The non-enzymatic antioxidant substances exhibited a highly enhanced capability. The pathogenesis-related protein (PR) genes were also highly up-regulated. In summary, we demonstrated that the interplay among MeJA plus CD and PQ may activate a complex signaling network to regulate plant defense mechanisms involving the up-regulation of detoxifying enzymes, induction of free-radical scavengers and overexpression of genes associated with plant defense pathways.