1H-NMR-based metabolomic profiling and proteomic analysis of soybean (Glycine max L.) in response to dicarboxylic acids (photon) application as a stress priming agent

Soybean (<i>Glycine</i> <i>max</i> L.) serves not only as food for humans, animals, and industrial purposes, but is also a plant that can be used to comprehend molecular mechanisms occurring in stress response to various development techniques. To reveal the effect of applying dicarboxylic acids as stress priming agents on a metabolic level in soybean leaf extracts, the chemical profile of methanolic extracts were collected at different time points (1 h, 2 h, 12 h, 24 h, 1 week, 2 weeks and 3 weeks) after spraying were analyzed using ¹H-NMR based metabolomics by way of PCA and OPLS-DA. The OPLS-DA revealed several metabolites, including organic acids (fumarate, citrate and malate) and amino acids (asparagine, alanine and GABA), which accumulated in higher amounts, with fumarate accumulating the highest in <i>Glycine</i> <i>max</i> L. leaf extracts compared to untreated leaves. Denaturing 1DE gels were prepared for MS-based protein analysis and the presence of fatty acids (linolenic, oleic and α-linolenic acid) were confirmed by gas chromatography coupled with mass spectrometry (GC-MS), which served as jasmonic acid precursors. The MS-based profiling of proteins on the denaturing 1DE gels revealed several proteins that were differentiated between the treated and untreated leaf extracts. These proteins included ferritins, CaM, ferredoxin-thioredoxin reductase and chalcone-flavanone isomerase 1A. Following the treatment, fumarate was significantly elevated at 12 h to 3 weeks, compared to other compounds. It is, therefore, proposed that elevated quantities of fumarate could be related to the KEAP1-NRF2 metabolic pathway. This study represents the initial investigation of the effect of dicarboxylic acid application as a stress priming agent on <i>Glycine</i> <i>max</i> L. using ¹H-NMR metabolomic analysis, GC-MS and proteomic analysis.