Rachel Alves Maia, Kátia Daniella da Cruz Saraiva, André Luiz Maia Roque, Karine Leitão Lima Thiers, Clesivan Pereira dos Santos, João Hermínio Martins da Silva, Daniel Ferreira Feijó, Birgit Arnholdt-Schmitt, José Hélio Costa

Plastid terminal oxidase (PTOX) is a chloroplast enzyme that catalyzes oxidation of plastoquinol (PQH₂ ) and reduction of molecular oxygen to water. Its function has been associated with carotenoid biosynthesis, chlororespiration and environmental stress responses in plants. In the majority of plant species, a single gene encodes the protein and little is known about events of PTOX  gene duplication and their implication to plant metabolism. Previously, two putative PTOX  (PTOX1  and 2 ) genes were identified in Glycine max , but the evolutionary origin and the specific function of each gene was not explored. Phylogenetic analyses revealed that this gene duplication occurred apparently during speciation involving the Glycine  genus ancestor, an event absent in all other available plant leguminous genomes. Gene expression evaluated by RT-qPCR and RNA-seq data revealed that both PTOX  genes are ubiquitously expressed in G. max  tissues, but their mRNA levels varied during development and stress conditions. In development, PTOX1  was predominant in young tissues, while PTOX2  was more expressed in aged tissues. Under stress conditions, the PTOX  transcripts varied according to stress severity, i.e., PTOX1  mRNA was prevalent under mild or moderate stresses while PTOX2  was predominant in drastic stresses. Despite the high identity between proteins (97%), molecular docking revealed that PTOX1 has higher affinity to substrate plastoquinol than PTOX2. Overall, our results indicate a functional relevance of this gene duplication in G. max  metabolism, whereas PTOX1  could be associated with chloroplast effectiveness and PTOX2  to senescence and/or apoptosis.