title: Deamination role of inducible glutamate dehydrogenase isoenzyme 7 in Brassica napus leaf protoplasts.
abstract: Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamate. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively and the isoenzyme profile in leaves is known to change on wounding. Here, parallels were sought between the effects of wounding and protoplast isolation because of the possible relevance of changes in GDH activity to the perturbed metabolism in recalcitrant B. napus protoplasts. When leaf protoplasts of B. napus were isolated, GDH7 isoforms predominated. Transcription of GDH2, which encodes the GDH α subunit, was activated and translation of the GDH2 mRNA was also activated to synthesize α subunit polypeptides. When detached leaves absorbed either acidic 5mM jasmonic acid or salicylic acid solutions via petioles, GDH7 isoenzymes were activated and the GDH isoenzyme patterns were similar to those of protoplasts. Salicylic acid β-glycosides were generated soon after treatment with the pectinase-cellulase enzyme solution and peaked at 1h. NMR spectroscopic analysis of protoplasts and unstressed leaves incubated with 5mM (15)NH(4)Cl showed that the change in GDH isoenzyme profile had no effect on ammonium assimilation. Protoplast isolation changed the redox state with NAD(P)H and oxidized glutathione levels increasing, and ascorbate, dehydroascorbate, NAD(P) and glutathione decreasing. ATP content in protoplasts declined to 2.6% of that in leaves, while that in wounded leaves increased by twofold. It is concluded that GDH7 does not support net amination in vivo and it is suggested that the increase in GDH7 activity is a response to oxidative stress during protoplast isolation.
authors.affiliation: Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8510, Japan. masamiwata@faculty.chiba-u.jp
authors.forename: Masami
authors.initials: M
authors.lastname: Watanabe
authors.forename: Ohnishi
authors.initials: O
authors.lastname: Yumi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Itoh
authors.forename: Kaori
authors.initials: K
authors.lastname: Yasuda
authors.forename: Kazunari
authors.initials: K
authors.lastname: Kamachi
authors.forename: R George
authors.initials: RG
authors.lastname: Ratcliffe
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000586:Amination; D029688:Brassica napus / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002952:Citric Acid Cycle / Q000187:drug effects; D003517:Cyclopentanes / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D054883:Oxylipins / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis; D018515:Plant Leaves / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D003517:Cyclopentanes; D007527:Isoenzymes; D054883:Oxylipins; D010446:Peptide Fragments; D000644:Quaternary Ammonium Compounds; C011006:jasmonic acid; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D020156:Salicylic Acid