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Plastid terminal oxidase (PTOX) is a chloroplast enzyme that catalyzes oxidation of plastoquinol (PQH 2 ) 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.

Plant plastoquinol oxidase (PTOX) is a chloroplast oxidoreductase involved in carotenoid biosynthesis, chlororespiration, and response to environmental stresses. The present study aimed to gain insight of the potential role of nucleotide/amino acid changes linked to environmental adaptation in PTOX gene/protein from Arabidopsis thaliana accessions. SNPs in the single-copy PTOX gene were identified in 1190 accessions of Arabidopsis using the Columbia-0 PTOX as a reference. The identified SNPs were correlated with geographical distribution of the accessions according to altitude, climate, and rainfall. Among the 32 identified SNPs in the coding region of the PTOX gene, 16 of these were characterized as non-synonymous SNPs (in which an AA is altered). A higher incidence of AA changes occurred in the mature protein at positions 78 (31%), 81 (31.4%), and 323 (49.9%). Three-dimensional structure prediction indicated that the AA change at position 323 (D323N) leads to a PTOX structure with the most favorable interaction with the substrate plastoquinol, when compared with the reference PTOX structure (Columbia-0). Molecular docking analysis suggested that the most favorable D323N PTOX-plastoquinol interaction is due to a better enzyme-substrate binding affinity. The molecular dynamics revealed that plastoquinol should be more stable in complex with D323N PTOX, likely due a restraint mechanism in this structure that stabilize plastoquinol inside of the reaction center. The integrated analysis made from accession geographical distribution and PTOX SNPs indicated that AA changes in PTOX are related to altitude and rainfall, potentially due to an adaptive positive environmental selection.