Molecular characterization and transcriptional pro�ling suggested structural and functional divergence in ascorbate peroxidase (APX) and APX-related (APX-R) genes in Triticum aestivum L.

Background: Ascorbate peroxidases (APXs) are heme-dependent H2O2 scavenging enzymes providing the antioxidant defense and regulating myriad biological processes. APX-related (APX-R) is an additional form of APX protein which lacks peroxidase activity but form dimer with the chloroplastic APX (chlAPX). Despite of tremendous their functions, a systematic and comparative study of these genes is limited in Triticum aestivum L. Results: Herein, a total of 21 TaAPX and six TaAPX-R genes were identi�ed from the A, B and D sub genomes of Triticum aestivum, which form nine homeologous groups. These identi�ed genes consisted of three pairs of paralogous genes which confronted pre-hybridization gene duplication under purifying selection. An unequal evolutionary rate after gene duplication suggested functional divergence between paralogous genes. The phylogenetic study classi�ed TaAPXs into four distinct clades, where TaAPX-R proteins were clustered in clade I. The members of each clade showed similarity in gene and protein architecture, motifs distribution, sub-cellular localization that suggested their conserved nature. The tertiary structure analysis of three TaAPXs and one TaAPX-R revealed the presence of helices and coils, and residues involved in ligand binding. Transcriptional pro�ling of TaAPX and TaAPX-R genes suggested their speci�c role during development and abiotic and biotic stress response. Modulated transcript expression and APX enzyme activity during heat, drought and salt stress con�rmed their role in these stress conditions. Co-expression analyses predicted interaction of these genes with the other genes involved in numerous biological processes. These genes were also found to be regulated by miRNAs associated with various developmental pathways and/or stress response. Further, they also exhibited interaction with the mediator compounds of Halliwell-Asada cycle. Conclusions: Overall, the present study reported numerous molecular and structural features of TaAPXs and TaAPX-Rs, and their role in plant development and stress response. It provides a platform for detailed functional characterization of TaAPX and TaAPX-R genes in future studies. albumin (BSA) was used as standard. The APX activity was determined by analyzing the decrease in absorbance of ascorbate at 290 nm with an absorbance coe�cient of 2.8 mM -1 cm -1 [78] using Eppendorf BioSpectrometer® kinetic. The assay was performed in 1 ml reaction volume consisting of 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA, and 0.1 mM hydrogen peroxide. The reaction was started by adding 10 µl of crude protein extract and the absorbance decrease was recorded for 3 minutes [78]. All the experiments were performed in triplicates and expressed as mean ± SD. The APX activity data for heat, drought and their combination was statistically analyzed by analysis of variance (ANOVA) at 5% probability level using SPSS software. The post hoc Tukey’s test was used to examine the signi�cant difference among the treatments (P <0.05). In case of salt stress, the fold change in APX activity was analyzed and expressed as mean ± SD.