OBJECTIVE: We previously identified AtWRKY50 as a transcriptional activator of SAR gene PR1. Although PR1 accumulates to high levels in plants after attack by pathogens, its function is still elusive. Here we investigated the effects of overexpression of several WRKY proteins, including AtWRKY50, on the metabolome of Arabidopsis thaliana.
METHODS: The influence of overexpression of WRKY proteins on the metabolites of Arabidopsis was investigated by using an NMR spectroscopy-based metabolomic approach. The 1H NMR data was analysed using the multivariate data analysis methods, such as principal component analysis, hierarchical cluster analysis and partial least square-discriminant analysis.
RESULTS: The results showed that the metabolome of transgenic Arabidopsis seedlings overexpressing AtWRKY50 was different from wild type Arabidopsis and transgenic Arabidopsis overexpressing other WRKY genes. Amongst other metabolites, sinapic acid and 1-O-sinapoyl-β-D-glucose especially appeared to be the most prominent discriminating metabolites, accumulating to levels 2 to 3 times higher in the AtWRKY50 overexpressor lines.
CONCLUSION: Our results indicate a possible involvement of AtWRKY50 in secondary metabolite production in Arabidopsis, in particular of hydroxycinnamates such as sinapic acid and 1-O-sinapoyl-β-D-glucose.
METHODS: The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan's post hoc test, and correlation among the different activities was performed by Pearson's correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts.
RESULTS: It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol:water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18 μg mL- 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50 μg mL- 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS.
CONCLUSION: For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential.