Table 1.
List of identified GST members in Medicago trancatula along with their detailed information and subcellular localization.
Fig 1.
Chromosomal distribution and duplication analysis of MtGST genes.
In the diagram, eight chromosomes and one scaffold region (Chr 0) are represented in partial circles with different colours. MtGST genes in different chromosomes are indicated by red labels. Different coloured lines connecting two chromosomal regions indicate the duplicated gene pairs in Medicago. The illustration was generated using CIRCOS software.
Table 2.
Duplicated GST genes and the probable dates of duplication in Medicago.
Fig 2.
Gene structure of MtGST with an evolutionary relationship.
The phylogenetic tree was generated using MEGA7. Exon-intron analyses of MtGST genes were created with GSDS tool. Lengths of introns and exons of MtGST genes were exhibited proportionally. Exons are shown as red boxes and introns are shown as black lines, while the untranslated region is shown as blue boxes.
Table 3.
Detailed information about the maximum possibility of glycation modification among MtGST proteins.
Fig 3.
Expression profiles of sixty-eight MtGST transcripts in different developmental stages and anatomical tissues.
(A) Expression of 68 MtGST genes was analyzed at five major anatomical tissues, such as primary cell, seedlings, inflorescence, shoot and roots. (B) Expression of the same 68 MtGST genes was analyzed at seven distinct developmental stages, such as germination, seedlings, main roots, axillary shoot, developed flower, flower and pods, and mature pods. Expression data was retrieved from genevestigator (https://genevestigator.com/gv/) and the heatmap was created with hierarchical clustering of Manhattan distance correlation using MeV software package. A colour scale is provided along with the heat map to recognize the differential pattern of expression.
Fig 4.
Expression pattern of MtGST genes under various abiotic and biotic stresses.
(A) Expression pattern of sixty-eight MtGST genes was analyzed in response to two devesting abiotic stresses- salt and drought. Expressions were analyzed at 6h, 24h and 48h shoot samples for salinity, and samples were analyzed at 2d, 3d, 4d, 7d, 10d, 14d and 14d+1d rewatering in both shoot and root tissues for drought. (B) The expression profile was also analyzed in response to two common fungal pathogens- Phymatotrichopsis omnivore and Macrophomina phaseolina, and four infectious gram-negative bacteria—Sinorhizobium meliloti, Ralstonia solanacearum, Salmonella enterica and Escherichia coli at various day after infection (dpi). Fold change in expression as compared to control was used to generate the heatmaps with hierarchical clustering of Manhattan distance correlation in MeV software package. The colour scale provided at the bottom of the figure represents the level of expression. The stress-induced upregulation or down-regulation of MtGST transcripts is indicated by the red or yellow colour, respectively.
Fig 5.
Analysis of cis-acting elements in the putative promoter of MtGSTs.
One kb 5′ upstream region of all the identified MtGST genes were retrieved and analyzed through the PlantCARE database to identify the presence and number of stress and hormone-responsive cis-acting regulatory elements. The number of identified motifs were plotted against a particular gene in a bar diagram. The abundance of different cis-regulatory elements on each of the promoter was represented with different colours.
Fig 6.
Relative normalized expression level of 10 selected MtGST genes in response to drought and salt stresses.
Transcript abundance of ten selected MtGST genes were analyzed in the WT Medicago plants in response to two devastating abiotic stresses (salinity and drought) as compared to the respective control samples for 24h and 48h. Data represent (A-T) here the mean normalized expression value ± SD (n = 6). The Student’s t-test analysis indicated a significant alteration in their expression level as compared with the respective controls (marked with different letters). (U) Heatmap showed the fold change in expression of the selected genes in response to salinity and drought where red colour indicates upregulation and blue indicates downregulation as compared to controls.
Fig 7.
Homology modelling and molecular docking of four highly stress-responsive proteins.
Four MtGST proteins (MtGSTU17, MtGSTF8, MtGSTZ2 and MtGSTT2) were used for the molecular docking with two substrate glutathione (GSH) and 1-Chloro-2,4-dinitrobenzene (CDNB). The first column represents the predicted 3D structure, the second and third column represents the 2D interaction of each protein with GSH and CDNB, respectively. The green, light green, pink, purple Orange and red spheres represent residues involved in the hydrophobic interactions, carbon-hydrogen bond interactions, Pi-alkyl interactions, Pi–cation interactions and unfavorable acceptor–acceptor interactions, respectively.
Table 4.
Predicted binding affinity (Kcal/mol) of selected MtGST proteins against reduced glutathione (GSH) and 1-chloro 2,4 dinitrobenzene (CDNB).
Table 5.
Distribution of GST family members in twenty-two different plants species.