Metabolic and Proteomic Perspectives of Augmentation of Nutritional Contents and Plant Defense in Vigna unguiculata

Abstract

The current study enlists metabolites of Alstonia scholaris with bioactivities, and the most active compound, 3-(1-methylpyrrolidin-2-yl) pyridine, was selected against Macrophomina phaseolina. Appraisal of the Alstonia metabolites identified the 3-(1-methylpyrrolidin-2-yl) pyridine as a bioactive compound which elevated vitamins and nutritional contents of Vigna unguiculata up to ≥18%, and other physiological parameters up to 28.9%. The bioactive compound (0.1%) upregulated key defense genes, shifted defense metabolism from salicylic acid to jasmonic acid, and induced glucanase enzymes for improved defenses. The structural studies categorized four glucanase-isozymes under beta-glycanases falling in (Trans) glycosidases with TIM beta/alpha-barrel fold. The study determined key-protein factors (Q9SAJ4) for elevated nutritional contents, along with its structural and functional mechanisms, as well as interactions with other loci. The nicotine-docked Q9SAJ4 protein showed a 200% elevated activity and interacted with AT1G79550.2, AT1G12900.1, AT1G13440.1, AT3G04120.1, and AT3G26650.1 loci to ramp up the metabolic processes. Furthermore, the study emphasizes the physiological mechanism involved in the enrichment of the nutritional contents of V. unguiculata. Metabolic studies concluded that increased melibiose and glucose 6-phosphate contents, accompanied by reduced trehalose (-0.9-fold), with sugar drifts to downstream pyruvate biosynthesis and acetyl Co-A metabolism mainly triggered nutritional contents. Hydrogen bonding at residues G.357, G.380, and G.381 docked nicotine with Q9SAJ4 and transformed its bilobed structure for easy exposure toward substrate molecules. The current study augments the nutritional value of edible stuff and supports agriculture-based country economies.

Keywords: Macrophomina phaseolina; chemical-protein docking; defense pathways; glucanase isozyme; nutrition metabolism; phosphoglycerate kinase 3; physicochemical analysis; plant protein modeling; protein active pockets; protein-protein interaction.

Figure 1

Pearson’s correlation coefficient values of each compound (01–22) with disease control are plotted in the form of horizontal bars, green bars for negative values, and red bars for positive correlation values. The extraction coefficient values are provided in the numeric form for all the compounds (A). Structure and value of Nuclear Magnetic Resonance (NMR) spectrometry (¹H, ¹³C) analysis of the most active compound, 3-(1-methylpyrrolidin-2-yl) pyridine (B). Percentage disease control against the concentration gradient (0.00%, 0.2%, 0.4%, and 0.16%) of the most active compound, 3-(1-methylpyrrolidin-2-yl) pyridine (C), * p < 0.5.

Figure 2

Nutritional analysis of Vigna unguiculata after the application of 3-(1-methylpyrrolidin-2-yl) pyridine. Values of niacin, pyridoxine, pantothenic acid, thiamine, riboflavin, and folic acid contents are provided in (A), while ascorbic acid, protein, fat, fiber, and carbohydrate contents are shown in (B). Data of nine physiological parameters are plotted as (Net photo.) net photosynthetic rate (µ mol CO₂ m⁻² s⁻¹) (C), (internal CO₂ con.) internal CO₂ concentration (ppm) (D), stomatal conductance (mol H₂O m⁻² s⁻¹) (E), transpiration rate (mmol H₂O m⁻² s⁻¹) (F), Fv/Fm (G), (CAT) catalase (mmol H₂O₂ decomposed g⁻¹ FM) (H), (POX) peroxidase (units g⁻¹ (FM)) (I), (SOD) superoxide dismutase (units g⁻¹ (FM)) (J), and proline (µ mol g⁻¹(FM) (K). The experiment was replicated thrice, and mean values of nutritional contents were calculated to construct the plots.

Figure 3

Screening of the most active protein (MAP) of Vigna unguiculata through principal component analysis (PCA) against the treatment of bioactive compound 3-(1-methylpyrrolidin-2-yl) pyridine. The X-axis coefficient represents the affinity of individual proteins with augmentation of nutritional contents; however, the coefficient value on Y-axis is the interrelation of proteins with disease control. A total of 44 differentially expressed proteins are mentioned in the matrix plot of cultivar Elite (A), CP1 (B), White Star (C), and SA-Dandy (D). The average behavior of protein species with reference to bioactivities in all the four Vigna cultivars (E). The bilobed structure of screened MAP phosphoglycerate kinase 3-Q9SAJ4 (F).

Figure 4

Tertiary and primary structure of the most active protein species, Q9SAJ4, showing its two constituting monomers ranging 9-400 residues for monomer 1, and 3-70 residues for monomer 2 (A). Similarity chart for protein Q9SAJ4 structure with the target protein model (B). Energy-distribution plot QMEAN4 score comparing with a non-redundant set calculated at Z-Score >2 (C).

Figure 5

The co-expression interaction of all the differentially expressed protein species in Vigna unguiculata (A). The evolutionary tree on the left side was constructed by using the Neighbor-Joining method. The optimal tree had the sum of branch length = 51.83488287, whereas the evolutionary distances were computed by using the Poisson correction method and were in the units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The dendrogram on the upper side was plotted by using the Maximum Likelihood method and the JTT matrix-based model. The tree had the highest log likelihood (-56253.26). The tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances, estimated by using a JTT model and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA X. The chemical protein docking was drawn between Q9SAJ4 and 3-(1-methylpyrrolidin-2-yl) pyridine (B). The close-up of the chemical protein docking with the details of interacting residues (C).

Figure 6

Expression analysis of defense genes in four Vigna cultivars (A). Heatmap showing the extent of upregulation or downregulation of defense genes after the application of bioactive compound (B), p < 0.5. Composite change in the expression of defense-related genes in Vigna plants after the application of bioactive compound (C). Interrelation among the defense-related metabolic pathways (salicylic acid, SA; jasmonic acid, JA; and phytochelatin biosynthesis, PCB) before and after the treatment of the bioactive compound (D).

Figure 7

Alteration in the expression of glucanase isozymes in each cultivar of V. unguiculata after the application of 3-(1-methylpyrrolidin-2-yl) pyridine (A). Dendrogram of four cultivars of Vigna unguiculata based on the differences in glucanases (B). Physiochemical characteristics of the glucanase isozymes (C). The percentage share of each amino acid in the composition of the glucanase isozymes (D). Three-dimensional models of glucanase isozymes (Glu01, Glu02, Gl03, and Gl04) homologated with PSI-Blast (E). Secondary structure and disorder were calculated with Psi-pred and Diso-pred for the construction of a hidden Markov model. The largest pocket detected is shown in wireframe mode, colored red. The ProQ2 quality assessment algorithm was used to determine the quality of protein structure. The evolutionary history was inferred by using the UPGMA method. The optimal tree was drawn to scale, with the same branch lengths as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed by using the Poisson correction method. All ambiguous positions were removed by the pairwise deletion method.

Figure 8

The evidence view showing the binding affinities of Q9SAJ4 domain with other biochemicals (A). Different line colors represent the types of evidence for the association, as shown in Table S6; Supplementary Data Set 1. The confidence view representing the strength of ligand associations (protein–protein, chemical–protein, and chemical–chemical interactions), using different weight lines (B). Stronger associations are shown by thicker lines. Protein–protein interactions are shown in blue, chemical–protein interactions in green, and interactions between chemicals in red. Actions view depicting the modes of action in different colors (C).

Figure 9

Quantification of the kinase activity of phosphoglycerate kinase 3 (PGK3) in four cultivars of Vigna unguiculata, after and before the application of bioactive compound nicotine, 3-(1-methylpyrrolidin-2-yl) pyridine (A). Percentage increase in the kinase activity of PGK3 due to the application of nicotine on four Vigna cultivars (B). Sugar metabolism of V. unguiculata cultivars after the application of 3-(1-methylpyrrolidin-2-yl) pyridine (C). Signaling of plant defense responses and Vigna plant defense factors after the treatment with 3-(1-methylpyrrolidin-2-yl) pyridine (D).

Figure 10

Role of phosphoglycerate kinase activity of Q9SAJ4 in the form of a chain of connective events and the elevation of kinase activity after the application of nicotine treatment.