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.

Combining elicitor treatment of chitosan, methyl jasmonate, and cyclodextrin to induce the generation of immune response bioactive peptides in peanut hairy root culture.

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1-4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and ß-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1-4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries.

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.

Enhancement of adaptive response in peanut hairy root by exogenous signalling molecules under cadmium stress.

Plants counteract Cd toxicity by activating cellular stress responses. The simultaneous exogenous application of methyl jasmonate (MeJA) and methyl-ß-cyclodextrin (CD) before Cd exposure improved the response of Arachis hypogaea hairy root culture to the unfavourable effects of Cd toxicity. At 24 h after elicitation, genes that encode key enzymes in the phenylpropanoid biosynthesis pathway (i.e., PAL and RS3) were up-regulated to 3.2- and 5.4-fold changes respectively, thereby inducing stilbene production. The up-regulation of genes that encode transcription factors (i.e., ERF1 and ERF6) significantly increased the expression of several genes (PR4A, PR5, PR10, and chitinase) that encode the pathogenesis-related (PR) proteins to 25.8-, 45-, 5- and 12.6-fold changes, respectively. The more dramatic up-regulation of PR protein-encoding genes demonstrated the significant role of defence proteins in plant protective mechanisms. The prolonged (i.e., 72-h) treatment with MeJA + CD_Cd triggered adaptive responses by substantially increasing the levels of antioxidants, stilbenes, and other phenolic substances. These findings suggest that the interaction between signalling elicitors (MeJA and CD) and Cd modulates a complex signalling network for plant defence system.

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.

LC-MS/MS profiles and interrelationships between the anti-inflammatory activity, total phenolic content and antioxidant potential of Kalasin 2 cultivar peanut sprout crude extract.

Peanut is a potent natural source of phytochemical compounds and is associated with human health. In the present study, we determined the biological activity and chemical constituents of peanuts germinated for 0-9days. The ungerminated seed coat exhibited the highest antioxidant potential, phenolic compound content and anti-inflammatory activity. During the germination process, on the first germination day, root extract showed the highest antioxidant potential, phenolic compound content and anti-inflammatory activity. The kernel exhibited a low phenolic compound content and antioxidant activity at the early stage of germination and significantly increased after 9days of germination. Resveratrol increased to 7.19±0.07µg/g dry weight on the second day of germination. LC-MS/MS showed a variety of phenolic compounds and stilbene derivatives in different parts of germinated peanut. These results suggest that the peanut sprout exerts high anti-inflammatory effects that may be related to the polyphenolic content and antioxidant properties.

The Arabidopsis thaliana MEK AtMKK6 activates the MAP kinase AtMPK13.

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase or MEK. The Arabidopsis thaliana genome encodes 10 MEKs but few of these have been shown directly to activate any of the 20 Arabidopsis MAP kinases. We show here that functional complementation of the cell lysis phenotype of a mutant yeast strain depends on the co-expression of the Arabidopsis MEK AtMKK6 and the MAP kinase AtMPK13. The kinase activity of AtMPK13 is stimulated in the presence of AtMKK6 in yeast cells. RT-PCR analysis showed the co-expression of these two genes in diverse plant tissues. These data show that AtMKK6 can functionally activate the MAP kinase AtMPK13.

The MAP kinase kinase NtMEK2 is involved in tobacco pollen germination.

The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45(Ntf4)) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45(Ntf4), is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45(Ntf4). In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss-of-function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45(Ntf4) and/or SIPK, are involved in pollen germination.

MAP kinase phosphorylation of plant profilin.

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.