Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin

Melina López-Meyer, Nancy L. Paiva

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Abstract

Vestitone reductase (VR; E.C. 1.1.1._) and isoflavone reductase (IFR; E.C. 1.3.1.45) are phytoalexin-specific enzymes involved in the biosynthesis of medicarpin in alfalfa (Medicago sativa L.). Immunoblot analysis of total soluble protein extracts indicated that these proteins constitutively accumulate in roots and nodules of alfalfa plants, and in alfalfa cell cultures. VR and IFR proteins are not present in healthy leaves but are rapidly synthesized upon fungal infection, and levels in cell cultures increase following elicitation. Immunolocalization studies in alfalfa organs at the cellular level indicated that VR accumulates in a narrow zone of leaf cells surrounding fungal lesions, in all cell types of mature and young roots, and in nodule meristem and lateral nodular tissues. Subcellular localization studies using immunogold labelling clearly indicated that VR is a cytosolic enzyme in alfalfa, with the majority of VR appearing free in cytosol. Association of VR with preserved endoplasmic reticulum (ER) membrane fragments was observed in several sections, but sucrose density gradient fractionation did not confirm the ER association. Although VR exhibits maximum activity in vitro in relatively acidic buffers (pH 6.0, approaching the pH of the vacuole), and the final product of the pathway (medicarpin-3-O-glucoside-6″-O-malonate) accumulates in the vacuole, no immunogold signal was observed in the vacuole. Subcellular analysis of CaMV35S::IFR transgenic tobacco indicated that IFR is cytosolic as well. These results clearly indicate that the late steps of pterocarpan biosynthesis occur in the plant cytosol. © 2002 Elsevier Science Ltd. All rights reserved.
Original languageAmerican English
Pages (from-to)15-30
Number of pages16
JournalPhysiological and Molecular Plant Pathology
DOIs
StatePublished - 1 Jan 2002
Externally publishedYes

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vestitone reductase
medicarpin
Medicago sativa
phytoalexins
isoflavones
alfalfa
biosynthesis
vacuoles
Enzymes
enzymes
Vacuoles
cytosol
endoplasmic reticulum
cell culture
Endoplasmic Reticulum
Cytosol
Plant Root Nodules
Pterocarpans
Cell Culture Techniques
lesions (plant)

Cite this

@article{654d31ea50184552bbb289d7c1f0bf62,
title = "Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin",
abstract = "Vestitone reductase (VR; E.C. 1.1.1._) and isoflavone reductase (IFR; E.C. 1.3.1.45) are phytoalexin-specific enzymes involved in the biosynthesis of medicarpin in alfalfa (Medicago sativa L.). Immunoblot analysis of total soluble protein extracts indicated that these proteins constitutively accumulate in roots and nodules of alfalfa plants, and in alfalfa cell cultures. VR and IFR proteins are not present in healthy leaves but are rapidly synthesized upon fungal infection, and levels in cell cultures increase following elicitation. Immunolocalization studies in alfalfa organs at the cellular level indicated that VR accumulates in a narrow zone of leaf cells surrounding fungal lesions, in all cell types of mature and young roots, and in nodule meristem and lateral nodular tissues. Subcellular localization studies using immunogold labelling clearly indicated that VR is a cytosolic enzyme in alfalfa, with the majority of VR appearing free in cytosol. Association of VR with preserved endoplasmic reticulum (ER) membrane fragments was observed in several sections, but sucrose density gradient fractionation did not confirm the ER association. Although VR exhibits maximum activity in vitro in relatively acidic buffers (pH 6.0, approaching the pH of the vacuole), and the final product of the pathway (medicarpin-3-O-glucoside-6″-O-malonate) accumulates in the vacuole, no immunogold signal was observed in the vacuole. Subcellular analysis of CaMV35S::IFR transgenic tobacco indicated that IFR is cytosolic as well. These results clearly indicate that the late steps of pterocarpan biosynthesis occur in the plant cytosol. {\circledC} 2002 Elsevier Science Ltd. All rights reserved.",
author = "Melina L{\'o}pez-Meyer and Paiva, {Nancy L.}",
year = "2002",
month = "1",
day = "1",
doi = "10.1016/S0885-5765(02)90413-3",
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T1 - Immunolocalization of vestitone reductase and isoflavone reductase, two enzymes involved in the biosynthesis of the phytoalexin medicarpin

AU - López-Meyer, Melina

AU - Paiva, Nancy L.

PY - 2002/1/1

Y1 - 2002/1/1

N2 - Vestitone reductase (VR; E.C. 1.1.1._) and isoflavone reductase (IFR; E.C. 1.3.1.45) are phytoalexin-specific enzymes involved in the biosynthesis of medicarpin in alfalfa (Medicago sativa L.). Immunoblot analysis of total soluble protein extracts indicated that these proteins constitutively accumulate in roots and nodules of alfalfa plants, and in alfalfa cell cultures. VR and IFR proteins are not present in healthy leaves but are rapidly synthesized upon fungal infection, and levels in cell cultures increase following elicitation. Immunolocalization studies in alfalfa organs at the cellular level indicated that VR accumulates in a narrow zone of leaf cells surrounding fungal lesions, in all cell types of mature and young roots, and in nodule meristem and lateral nodular tissues. Subcellular localization studies using immunogold labelling clearly indicated that VR is a cytosolic enzyme in alfalfa, with the majority of VR appearing free in cytosol. Association of VR with preserved endoplasmic reticulum (ER) membrane fragments was observed in several sections, but sucrose density gradient fractionation did not confirm the ER association. Although VR exhibits maximum activity in vitro in relatively acidic buffers (pH 6.0, approaching the pH of the vacuole), and the final product of the pathway (medicarpin-3-O-glucoside-6″-O-malonate) accumulates in the vacuole, no immunogold signal was observed in the vacuole. Subcellular analysis of CaMV35S::IFR transgenic tobacco indicated that IFR is cytosolic as well. These results clearly indicate that the late steps of pterocarpan biosynthesis occur in the plant cytosol. © 2002 Elsevier Science Ltd. All rights reserved.

AB - Vestitone reductase (VR; E.C. 1.1.1._) and isoflavone reductase (IFR; E.C. 1.3.1.45) are phytoalexin-specific enzymes involved in the biosynthesis of medicarpin in alfalfa (Medicago sativa L.). Immunoblot analysis of total soluble protein extracts indicated that these proteins constitutively accumulate in roots and nodules of alfalfa plants, and in alfalfa cell cultures. VR and IFR proteins are not present in healthy leaves but are rapidly synthesized upon fungal infection, and levels in cell cultures increase following elicitation. Immunolocalization studies in alfalfa organs at the cellular level indicated that VR accumulates in a narrow zone of leaf cells surrounding fungal lesions, in all cell types of mature and young roots, and in nodule meristem and lateral nodular tissues. Subcellular localization studies using immunogold labelling clearly indicated that VR is a cytosolic enzyme in alfalfa, with the majority of VR appearing free in cytosol. Association of VR with preserved endoplasmic reticulum (ER) membrane fragments was observed in several sections, but sucrose density gradient fractionation did not confirm the ER association. Although VR exhibits maximum activity in vitro in relatively acidic buffers (pH 6.0, approaching the pH of the vacuole), and the final product of the pathway (medicarpin-3-O-glucoside-6″-O-malonate) accumulates in the vacuole, no immunogold signal was observed in the vacuole. Subcellular analysis of CaMV35S::IFR transgenic tobacco indicated that IFR is cytosolic as well. These results clearly indicate that the late steps of pterocarpan biosynthesis occur in the plant cytosol. © 2002 Elsevier Science Ltd. All rights reserved.

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DO - 10.1016/S0885-5765(02)90413-3

M3 - Article

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JO - Physiological and Molecular Plant Pathology

JF - Physiological and Molecular Plant Pathology

SN - 0885-5765

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