Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-04, Vol.91 (9), p.3867-3871
• Main Authors: Pineiro, M, Garcia-Olmedo, F, Diaz, I
• Format: Article
• Language: English
• Subjects: ACILTRANSFERASA; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ACYLTRANSFERASE; ADN RECOMBINADO; ADN RECOMBINE; Animal social behavior; Bacteria; Biological and medical sciences; Biology; Botany; Cells, Cultured; COMPOSE ORGANOSOUFRE; COMPUESTO ORGANICO DEL AZUFRE; Cysteine; Disulfides; Dithiothreitol - pharmacology; Enzymes; EXPRESION GENICA; EXPRESSION DES GENES; FOSFOTRASFERASA; Fundamental and applied biological sciences. Psychology; GENE; Gene expression; Gene Expression Regulation, Bacterial; GENES; GENETICA; GENETIQUE; GLICOSIDASAS; GLYCOSIDASE; In Vitro Techniques; Kanamycin Kinase; Messenger RNA; Molecular and cellular biology; Molecular genetics; NEOMICINA; NEOMYCINE; Nicotiana; NICOTIANA TABACUM; Oxidation-Reduction; PHOSPHOTRANSFERASE; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Plants, Toxic; Plasmids; POTENCIAL REDOX; POTENTIEL REDOX; Promoter Regions, Genetic; PROTOPLASTE; PROTOPLASTOS; Protoplasts; Recombinant Proteins - chemistry; RNA; RNA, Messenger - genetics; THIOL; TIOLES; Transcription, Genetic - drug effects; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Viability
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.9.3867

Activity of neomycin phosphotransferase II (NFTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NFTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol