Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products

Abstract

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.

Homologs of old yellow enzyme in plants

“…Recently, two members of the OPR gene family (OPR subgroup I) have been cloned and characterized in barley (Abu-Romman, 2012; Al-Momany and Abu-Romman, 2014). In peas, six members of just one OPR gene subgroup (OPRI subgroup) have been cloned and biochemically characterized, suggesting that the pea genome encodes more than six OPR genes (Matsui et al, 2004) (Figure 1).…”

“…AtOPR1, AtOPR2, pea PsOPR enzymes PsOPR1 to PsOPR6, rice OsOPR1, and tomato SlOPR1 and SlOPR2 belong to subgroup I. These enzymes cannot catalyze this step and therefore, are not a part of the JA biosynthetic pathway (Schaller and Weiler, 1997b;Schaller et al, 1998;Biesgen and Weiler, 1999;Sobajima et al, 2003;Matsui et al, 2004).…”

“…Plant OPRs are usually encoded by a multigene family. To date, several OPR isozymes have been identified in various plant species (Strassner et al, 1999;Sanders et al, 2000;Schaller et al, 2000;Stintzi and Browse, 2000;Strassner et al, 2002;Agrawal et al, 2003;Matsui et al, 2004;Zhang et al, 2005). Early biochemical studies of the enzymatic activity of OPRs in Arabidopsis and tomato have led to their classification into two subgroups (OPRI and OPRII), based on their differential preferences for 12-oxophytodienoate (OPDA) stereoisomers (Schaller et al, 1998;Strassner et al, 1999;Schaller et al, 2000).…”

Phylogenetic analysis, structural evolution and functional divergence of the 12-oxo-phytodienoate acid reductase gene family in plants

“…The first member of the OPR family in higher plants was identified from Arabidopsis thaliana , and named AtOPR1 [ 10 ]. Subsequently, other OPR genes were identified in the tomato [ 11 , 12 ], pea [ 13 ], rice [ 14 - 16 ] and maize [ 17 ] genomes. Earlier studies on the enzymatic activity of OPRs in Arabidopsis and tomato revealed that these enzymes have distinct substrate preferences and therefore have been classified into two groups, group I and II, depending on their substrate specificity [ 10 , 12 , 18 , 19 ].…”

Identificação e expressão de genes da biossíntese do jasmonato na interação entre Theobroma cacao e Moniliophthora perniciosa

“…A partir dos agrupamentos obtidos pela análise filogenética, foram desenhados oligonucleotídeos específicos para as seqüências dos genes identificados relacionados à via de biossíntese de JA e demais genes de interesse (Tabela 1 (Matsui et al, 2004).…”