TY - JOUR
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.
N1 - Funding Information:
Funding for this work was provided by The Samuel Roberts Noble Foundation, Ardmore, OK. The electron micrographs were generated at the Samuel Roberts Noble EM facility of the University of Oklahoma, Norman, OK with the generous assistance of Scott Russell and Gregory Strout. We thank Lining Guo for preparing the VR protein for antibody production, Xin Shun Ding, Bonnie Watson, Elison Blancaflor and Yiming Bao for helpful technical suggestions and confocal microscopy assistance, Susanne Rasmussen and Yiming Bao for careful review of the manuscript, and Darla Boydston and Broderick Stearns for their assistance with the photographic work.
PY - 2002
Y1 - 2002
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.
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.
KW - Alfalfa
KW - Immunolocalization
KW - Isoflavonoid biosynthesis
KW - Medicago sativa
KW - Medicarpin
KW - Phytoalexin
UR - http://www.scopus.com/inward/record.url?scp=0036424956&partnerID=8YFLogxK
U2 - 10.1016/S0885-5765(02)90413-3
DO - 10.1016/S0885-5765(02)90413-3
M3 - Artículo
SN - 0885-5765
VL - 61
SP - 15
EP - 30
JO - Physiological and Molecular Plant Pathology
JF - Physiological and Molecular Plant Pathology
IS - 1
ER -