The effect of drought stress on enzymatic and molecular changes of some antioxidants in parental and mutant bread wheat genotype using RNAseq. data

Wheat (Triticum aestivum L.) is one of the most important grains used in the world and its production is reduced in different regions due to drought stress. the plant antioxidant system can scavenge the reactive oxygen species (ROS) produced under drought. Induction of mutation using gamma ray is one of the common methods for genetic modification and identification of tolerant and resistant mutants. Mutant T65-58-8 is one of these drought tolerant genotypes that has been obtained by irradiation to Tabasi wheat genotype. The wheat plant needs irrigation during the flowering stage and drought stress is very important in this stage. In this study, in the flowering stage, the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) involved in the mechanisms of tolerance to oxidative damage of ROS in the flag leaf were studied and the expression of the genes related to these enzymes was investigated using RNAseq. method.

Drought stress was applied based on field capacity (FC). The experiment was performed as a factorial experiment in a completely randomized design with three replications. Factors studied in this experiment include genotype at two levels (Tabasi parent and Mutant T65-58-8) and drought stress at 5 levels (100% FC or control, 75%, 22%, re-sampling from control pots at 18% FC and sampling after re-irrigation to the pot when had 90% FC). Wheat growth stages can be studied by Zadoks index. SOD activity was measured by Minami and Yoshikawa method, CAT activity by Aebi method, GR activity by Foyer and Halliwell method and GPX activity by Hopkins and Tudhope method. RNA sequencing was performed using Illumina NovaSeq 6000. Gene expression was obtained based on sequencing data by Bowtie2, Tophat2, HTseq-count and Featurecount softwares. After normalization by generating FPKM, Log2FC gene expression was calculated.

Examination of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) indices showed a significant difference (p<0.01). Comparisons showed that SOD and CAT activity was higher in the mutant genotype at all stress levels. In the Tabasi genotype, re-irrigation increased the activity of SOD and CAT enzymes compared to the activity of this enzyme in field capacity 18%. In the study of GR and GPX enzymes, it was found that in both genotypes, the activity of this enzymes ancreases with increasing drought stress. In the Tabasi genotype, irrigation reduced the activity of GR enzyme and in the Tabasi and mutant genotypes, re-irrigation did not reduce the activity of GPX enzyme. Examination of GPX activity showed that this enzyme has a constant activity in mutant genotype under drought stress conditions. Examination of 21 genes related to SOD enzymes showed that six genes named FSD3(1), CSD1(2), FSD2(2) and CCS(1) mutant genotype had significant expression changes in drought stress conditions compared to the control. 6 catalase-related CAT1(3) and CAT2(3) genes from 14 genes related to CAT also had a significant increase in mutant genotype under drought stress compared to the control. One CAT1 gene and two CAT2 genes in mutant genotype had more expression in drought conditions than Tabasi genotype. 7 genes called CSA associated with GPX from 16 genes related to GPX also had a significant increase in mutant genotype under drought stress compared to the control. H2O2, KCN, chloroform-ethanol, sodium azide (NaN3), metal ions, carbohydrates, polyethylene glycol affect the activity of SOD. NaN3, amines, potassium cyanide and salicylic acid affect the activity of CAT. Gold, vitamin-E and selenium can also affect GPX activity.

The study shows that the activity of SOD, CAT and GPX enzymes in both genotypes has increased under drought stress conditions. However, comparison of the expression of known genes related to these three enzymes shows that only some of these known genes have been altered. One FSD3 gene, two CSD1 genes, two FSD2 genes and one CCS gene had significant expression changes in the mutant genotype under drought stress compared to the control. This could be considered as confirmation of the increase in superoxide dismutase activity in the mutant genotype under drought stress, but the difference in expression of these 6 genes in the mutant genotype under drought stress compared to the Tabasi genotype under drought stress was not significant. This indicates that other factors in the mutant genotype under drought stress have increased the activity of SOD enzyme or limited the production or activity of this enzyme in the Tabasi genotype under drought stress. from the 6 altered genes expressed in the mutant genotype under drought stress compared to the control, one gene called CAT1 and two genes called CAT2 in the mutant genotype under drought stress showed more expression than the Tabasi genotype under drought stress. These genes can be introduced as genes involved in more tolerance of mutant genotype to drought stress than Tabasi genotype under drought stress. 7 genes called CSA had a significant change in expression in the mutant genotype under drought stress compared to the control, but these genes were not significantly different in the mutant genotype compared to the Tabasi under drought stress. Comparison of glutathione reductase enzyme in mutant and Tabasi genotypes in drought condition showed that the activity of this enzyme in the two genotypes was not significantly different. The activity of SOD, CAT and GPX enzymes can be inhibited or intensified by chemical agents and compounds.