Molecular cloning, characterization and expression analysis of two 12-oxophytodienoate reductases (NtOPR1 and NtOPR2) from Nicotiana tabacum.

Abstract

12-oxophytodienoic acid (OPDA) is a signaling molecule involved in defense and stress responses in plants. 12-oxophytodienoate reductase (OPR) is involved in the biosynthesis of jasmonic acid and trigger the conversion of OPDA into 3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). Sequence analysis revealed that Nicotiana tabacum 12-oxophytodienoate reductase 1 (OPR1) and OPR2 encoded polypeptides of 375 and 349 amino acids with molecular masses of 41.67 and 39.04 kilodaltons (kDa), respectively, while the deduced protein sequences of NtOPR1 and NtOPR2 showed high homology with other 12-oxophytodienoate reductases. BLAST (Basic local alignment search tool) analysis revealed that both NtOPRs belong to the family of Old Yellow Enzymes (OYE), and analysis of genomic DNA structure indicated that both genes include 5 exons and 4 introns. Phylogenetic analysis using MEGA X showed that NtOPR1 and NtOPR2 shared a close evolutionary relationship with Nicotiana attenuata 12-oxophytodienoate reductases. In silico analysis of subcellular localization indicated the probable locations of NtOPR1 and NtOPR2 to be the cytoplasm and the peroxisome, respectively. Tissue-specific expression assays via qRT-PCR revealed that NtOPR1 and NtOPR2 genes were highly expressed in Nicotiana tabacum roots, temperately expressed in leaves and flowers, while low expression was observed in stem tissue. Presently, two 12-oxophytodienoate reductase genes (NtOPR1 and NtOPR2) were cloned and comprehensively characterized. Our findings provide comprehensive analyses that may guide future deep molecular studies of 12-oxophytodienoate reductases in Nicotiana tabacum.