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

Hidenori Matsui, G. Nakamura, Y. Ishiga, H. Toshima, Y. Inagaki, Kazuhiro Toyoda, T. Shiraishi, Yuki Ichinose

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalMolecular Genetics and Genomics
Volume271
Issue number1
DOIs
Publication statusPublished - Feb 2004

Fingerprint

Peas
Oxidoreductases
Acids
Genes
Reverse Transcriptase Polymerase Chain Reaction
Fabaceae
12-oxophytodienoate reductase
Complementary DNA
Clone Cells
Genomic Library
Gene Library
Transcriptome
Introns
Amino Acid Sequence
Exons
Proteins

Keywords

  • Coronatine
  • Flavoproteins
  • Jasmonic acid
  • Oxophytodienoic acid reductase (OPR)
  • Suppressor

ASJC Scopus subject areas

  • Genetics

Cite this

@article{2078f0a9faa847c4af1ea64183db7af6,
title = "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.",
keywords = "Coronatine, Flavoproteins, Jasmonic acid, Oxophytodienoic acid reductase (OPR), Suppressor",
author = "Hidenori Matsui and G. Nakamura and Y. Ishiga and H. Toshima and Y. Inagaki and Kazuhiro Toyoda and T. Shiraishi and Yuki Ichinose",
year = "2004",
month = "2",
doi = "10.1007/s00438-003-0948-6",
language = "English",
volume = "271",
pages = "1--10",
journal = "Molecular Genetics and Genomics",
issn = "1617-4615",
publisher = "Springer Verlag",
number = "1",

}

TY - JOUR

T1 - Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products

AU - Matsui, Hidenori

AU - Nakamura, G.

AU - Ishiga, Y.

AU - Toshima, H.

AU - Inagaki, Y.

AU - Toyoda, Kazuhiro

AU - Shiraishi, T.

AU - Ichinose, Yuki

PY - 2004/2

Y1 - 2004/2

N2 - 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.

AB - 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.

KW - Coronatine

KW - Flavoproteins

KW - Jasmonic acid

KW - Oxophytodienoic acid reductase (OPR)

KW - Suppressor

UR - http://www.scopus.com/inward/record.url?scp=1542284551&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1542284551&partnerID=8YFLogxK

U2 - 10.1007/s00438-003-0948-6

DO - 10.1007/s00438-003-0948-6

M3 - Article

C2 - 14727182

AN - SCOPUS:1542284551

VL - 271

SP - 1

EP - 10

JO - Molecular Genetics and Genomics

JF - Molecular Genetics and Genomics

SN - 1617-4615

IS - 1

ER -