Phylogenetic Analysis Of OPR Gene Family In Plants And Study Of Molecular Biological Functions Of OPR Family Genes In Rice

Jasmonic acid (JA) is a plant hormone and exists widely in plant, which is derived from linolenic acid via the octadecanoid pathway. Moreover, JA not only acts as plant growth regulator in various developmental processes, but also is local and systematic signaling molecule to response several of biotic and abiotic stresses. The 12-oxo-phytodienoic acid reductase (OPR) is one of the key enzymes in the octadecanoid pathway, which controls the last step of JA biosynthesis. In plants, the OPR genes, which belong to the old yellow enzyme (OYE) family, are flavin mononucleotide (FMN)-dependent oxidoreductases and form multigene families. Although discoveries about this family in Arabidopsis and other species have been reported in some studies, the evolution and biological functions (i.e. biochemical and physiological functions) of multiple OPRs in plants are not clearly understood. Therefore, based on the previous study of S5 on chromosme 6 of rice in our lab, we take the strategies of combining comparative genomics with the experimental methods and techniques of modern molecular biology, to investigate the phylogenetic evolution of OPR gene family in plants, and the protein structures, biochemical & physiological functions of OPR family genes in rice.Firstly, a comparative genomic analysis was performed by using a comprehensive bioinformatics & phylogenetic approaches to investigate the phylogenetic relationship, structural evolution and functional divergence among OPR paralogues in plants. The main results were as following:1) By using public database resources, 105 OPR genes were identified from plant genomes, including 74 OPR genes from 11 species representing the 6 major green plant lineages: green algae (Chlamydomonas reinhardtii and Volvox carteri), mosses (Physcomitrella patens), lycophytes (Selaginella moellendorffii), gymnosperms (Picea sitchensis), monocots (Oryza sativa, Sorghum bicolor and Zea mays) and dicots (Arabidopsis thaliana, Populus trichocarpa and Medicago truncatula).2) Phylogenetic analysis showed that seven well-conserved subfamilies exist in plants. All OPR genes from green algae were clustered into a single subfamily i.e. sub.Ⅶ, while those from land plants fell into other six subfamilies i.e. subs.Ⅰ~Ⅵ. Further analysis revealed that lineage-specific expansion, especially by tandem duplication, contributed to the current OPR subfamilies in land plants after divergence from aquatic plants.3) Interestingly, exon/intron structure analysis showed that the gene structures of OPR paralogues exhibits diversity in intron number and length, while the intron positions and phase were highly conserved across different lineage species. These observations together with the phylogenetic tree revealed that successive single intron loss, as well as indels within introns, occurred during the process of structural evolution of OPR paralogues. Then an evolution model was constructed to explain for the structural evolution from ancestral OPR to current OPR genes in plant species of different lineages.4) Functional divergence analysis revealed that altered functional constraints have occurred at specific amino acid positions after diversification of the paralogues, which leaded to significant functional divergence among all OPR subfamilies and their members. Strikingly, analysis of the site-specific profiles established by posterior probability revealed that the positive-selection sites and/or critical amino acid residues for functional divergence are mainly distributed inα-helices and substrate binding loop, indicating the functional importance of these regions for this protein family.Secondly, based on the phylogenetic analysis of OPR gene family in plant, five OsOPR genes (OsOPR04-1/08-1/06-1/01-1/02-1), representing five subfamilies (subs.Ⅰ~Ⅴ), were selected; then a comparative study was performed by using homology modeling, prokaryotic expression, semi-quantitative RT-PCR and transgenic techniques to provide insights into the five OsOPR protein structures, biochemical properties and physiological importance in rice. The main results were as following:1) Comparative analysis of the three-dimensional (3D) structure by homology modeling indicated all five OsOPR proteins contained a highly conserved backbone and consisted of (α/β)8-barrels. Notably, two middle variable regions (MVRⅰandⅱ) were also detected and defined. MVRⅰis comprised of substrate and protein, and MVRⅱaffects the substrate entrance into a "pocket" formed by MVRⅰ.2) Analysis of enzymatic characteristics revealed that all five OsOPR fusion proteins exhibit distinct substrate specificity. Different catalytic activity was observed using OPDA, trans-2-hexen-1-al and maleic acid as substrates, suggesting OsOPR family genes participate in two main branches of the octadecanoid pathway, including the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) pathways, and the tricarboxylic acid cycle (TCA) energy metabolism pathway that regulates various developmental processes and/or defense responses.3) The transcript profiles of five OsOPR genes exhibited strong tissue-specific and inducible expression patterns under abiotic stress, hormones and plant wounding treatments. Furthermore, OsOPR04-1 and OsOPR08-1 (subs.ⅠandⅡ, respectively) transcripts were observed in all selected tissues and with all above-stress treatments, suggesting these two subfamilies play an important role during different developmental stages and in response to stresses. OsOPR06-1, OsOPR01-1 and OsOPR02-1 (subs.Ⅲ,ⅣandⅤ, respectively) expression were strongly up-regulated with hormone treatments in roots, which suggested these three subfamilies play an important role in responding to hormones especially ABA and IAA signals in roots.4) Based on the results from structure comparison, enzymatic characteristics and expression profiles, a model was proposed to elucidate the functions of OsOPR subfamilies. This study highlights important implications in the search for the true physiological role of OPR family members. Further experimental verification of these findings such as reverse genetics (gene over-expression or gene interference) may provide valuable information on the OPRs'physiological functions.