Caracterização, análise filogenética e perfil de expressão dos genes do ciclo ascorbato-glutationa de soja (Glycine max)

Abstract

Under normal conditions, hydrogen peroxide (H2O2) plays a key role as a secondary messenger in response to different cellular processes. However, environmental stresses often lead to H2O2 overproduction causing oxidative damage in plants. Consequently, such deleterious effects often result in low yield or even plant death. In order to mitigate the harmful effects of H2O2, plants have a set of enzymes belonging to the ascorbate-glutathione cycle named ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1), and glutathione reductase (GR; EC 1.6.4.2). The aim of this study was to characterize, analyze the phylogeny and expression profile of multigene families encoding APX, MDHAR, DHAR, and GR in soybean (Glycine max) during stress conditions and development. Initially, the genes coding for the ascorbate-glutathione cycle were searched through of soybean genome, nine other leguminous species, Arabidopsis thaliana (reference) and the Volvox carteri algae (outgroup) deposited in GenBank. Next, the deduced protein sequences were used for phylogenetic analysis through the MEGA 7 program. In silico expression analyses of the genes encoding the ascorbate-glutathione cycle of soybeans were conducted using transcriptome data deposited in GenBank. The results showed that the ascorbate-glutathione cycle is encoded by 24 genes in both G. max and G. soja, while a maximum of 15 genes were found in the other leguminous species. Phylogenetic analysis revealed that the APX, MDHAR, DHAR, and GR proteins are formed by five, three, two and two clades, respectively. Then, proteins of each clade were classified according to those previously established for Arabidopsis thaliana. For G. max, proteins were named Gm-APX-1a, 1b, 2, 3a, 3b, 4a, 4b, 5a1, 5a2, 5b1, and 5b2; Gm-MDHAR-1, 2a, 2b, 3a, and 3b; Gm-DHAR-1a, 1b, 2a, and 2b; Gm-GR-1a, 1b, 2a, and 2b. In silico expression analyses of G. max genes submitted to stress and development conditions revealed that all genes are functional (except for Gm-APX-5a2), however, they exhibit characteristic expression profiles of tissue, stress and stage of development. The number of Gm-APX-3a, Gm-GR-1a, Gm-MDHAR-2b and Gm-MDHAR-3b transcripts, encoding chloroplast, cytosol, peroxisomes and cytosol proteins, respectively, increased under conditions of high salinity, dry, high ethylene concentration, and attack of the Fusarium oxysporum fungus. In addition, promoter regions of Gm-APX-3a and Gm-MDHAR-3b genes presented cis MYB elements responsive to water deficit while promoter region of the Gm-MDHAR-2b gene has a large amount of GT1 elements responsive to salinity. The results suggest these genes play an important role in antioxidant protection in soybeans submitted to stress. While different expression profiles for cotyledon and seed development suggest an important involvement Gm-APX-4a, Gm-APX-4b, Gm-MDHAR-3a, Gm-MDHAR-3b, Gm-GR-1a and, Gm-GR-1b and Gm-APX-5b1, Gm-MDHAR-2a, and Gm-MDHAR-2b genes (encoding cytosolic and peroxissomal proteins, respectively), root developmental zones showed genes that encoded cytosolic (Gm-APX-4b, Gm-MDHAR-3a, Gm-MDHAR-3b, Gm-DHAR-1a, Gm-GR-1a, and Gm-GR-1b), chloroplastidial (Gm-APX-2), and peroxisomal proteins (Gm-APX- 5a1 and Gm-APX-5b2) positively expressed. The present study reveal the diversity of genes and transcriptional profiles of APX, MDHAR, DHAR and GR families in soybean and suggest the complementation of antioxidant defense in different cellular compartments during development and stress.