Identification and Comparative Analysis of H2O2-Scavenging Enzymes (Ascorbate Peroxidase and Glutathione Peroxidase) in Selected Plants Employing Bioinformatics Approaches

Abstract

Among major reactive oxygen species (ROS), hydrogen peroxide (H2O2) exhibits dual roles in plant metabolism. Low levels of H2O2 modulate many biological/physiological processes in plants; whereas, its high level can cause damage to cell structures, having severe consequences. Thus, steady-state level of cellular H2O2 must be tightly regulated. Glutathione peroxidases (GPX) and ascorbate peroxidase (APX) are two major ROS-scavenging enzymes which catalyze the reduction of H2O2 in order to prevent potential H2O2-derived cellular damage. Employing bioinformatics approaches, this study presents a comparative evaluation of both GPX and APX in 18 different plant species, and provides valuable insights into the nature and complex regulation of these enzymes. Herein, (a) potential GPX and APX genes/proteins from 18 different plant species were identified, (b) their exon/intron organization were analyzed, (c) detailed information about their physicochemical properties were provided, (d) conserved motif signatures of GPX and APX were identified, (e) their phylogenetic trees and 3D models were constructed, (f) protein-protein interaction networks were generated, and finally (g) GPX and APX gene expression profiles were analyzed. Study outcomes enlightened GPX and APX as major H2O2-scavenging enzymes at their structural and functional levels, which could be used in future studies in the current direction.

Keywords: ROS; antioxidant; chloroplast; mitochondria; peroxisome; signal transduction.

Figure 1

Phylogenetic tree of glutathione peroxidase (GPX) homologs from 18 plant species. Tree was constructed by MEGA 6 using Maximum likelihood (ML) method with 1000 bootstraps. Segment classification based on the consensus of two subcellular localization servers, CELLO and WoLF PSORT as well as tree topology for ambiguous sequences. Red segment includes cytosolic, extra cellular, and plasma membrane related GPXs, green segment contains mitochondrial and chloroplast related GPXs, blue segment only have cytosolic GPXs, cyan segment includes cytosolic, and chloroplast/mitochondrial related GPXs, yellow segment contains cytosolic/nuclear related GPXs, and non-colored segment has lower plant Chlamydomonas GPX with chloroplastic/mitochondrial relation.

Figure 2

Expression profile of Arabidopsis glutathione peroxidase GPX1-8 genes at 105 anatomical parts (A) and 10 developmental stage levels (B). Genes and conditions with similar profiles were comparatively analyzed using hierarchical clustering tool with Euclidean distance method at Genevestigator platform.

Figure 3

3D models of predicted Arabidopsis glutathione peroxidase GPX1-8 sequences. Models were constructed by using Phyre² server for AT2G25080.1 (GPX1), AT2G31570.1 (GPX2), AT2G43350.1 (GPX3), AT2G48150.1 (GPX4), AT3G63080.1 (GPX5), AT4G11600.1 (GPX6), AT4G31870.1 (GPX7), and AT1G63460.1 (GPX8) sequences, and colored by rainbow from N- to C-terminus.

Figure 4

Predicted 10 interaction partners of Arabidopsis cytosolic glutathione peroxidase GPX2. Interactome was generated using cytoscape with STRING data. APX1 (L-ascorbate peroxidase), GSH2 (glutathione synthetase), GSTF6 (glutathione S-transferase F6), GSTT1 (glutathione S-transferase THETA 1), PER1 (1-Cys peroxiredoxin PER1), AT1G65820 (glutathione S-transferase), GSTF12 (glutathione S-transferase phi 12), GSTF2 (glutathione S-transferase F2), GSTF8 (glutathione S-transferase F8), and GSTU19 (glutathione S-transferase U19) proteins were predicted as main interaction partners of Arabidopsis cytosolic GPX2.

Figure 5

Phylogenetic tree of ascorbate peroxidase (APX) homologs from 18 plant species. Tree was constructed by MEGA 6 using Maximum likelihood (ML) method with 1000 bootstraps. Segment classification based on the consensus of two subcellular localization servers, CELLO and WoLF PSORT as well as tree topology for ambiguous sequences. Blue, red, and green segments include chloroplast/mitochondrial related APXs with mainly longer, medium and short sequences, respectively, while cyan and yellow segments contain longer and shorter cytosolic APX sequences, respectively.

Figure 6

Expression profile of Arabidopsis ascorbate peroxidase APX1-6, TAPX and SAPX genes at 105 anatomical parts (A) and 10 developmental stage levels (B). Genes and conditions with similar profiles were comparatively analyzed using hierarchical clustering tool with Euclidean distance method at Genevestigator platform.

Figure 7

3D models of predicted Arabidopsis ascorbate peroxidase APX1-6, APXT, and APXS sequences. Models were constructed by using Phyre² server for AT1G07890.1 (APX1), AT3G09640.1 (APX2), AT4G35000.1 (APX3), AT4G09010.1 (APX4), AT4G35970.1 (APX5), AT4G32320.1 (APX6), AT1G77490.1 (APXT), and AT4G08390.1 (APXS) sequences, and colored by rainbow from N- to C-terminus.

Figure 8

Predicted 10 interaction partners of Arabidopsis cytosolic ascorbate peroxidase APX1. Interactome was generated using cytoscape with STRING data. MDHAR (monodehydroascorbate reductase), GPX2 (glutathione peroxidase 2), DHAR1 (dehydroascorbate reductase), MDAR1 (monodehydroascorbate reductase 1), RHL41 (zinc finger protein ZAT12), ATPQ (ATP synthase subunit d), FBP (fructose-1,6-bisphosphatase), ATMDAR2 (monodehydroascorbate reductase (NADH)), CYTC-1 (cytochrome c-1), and CYTC-2 (cytochrome c-2) proteins were predicted as main interaction partners of Arabidopsis cytosolic APX1.