Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.
Activity of neomycin phosphotransferase II (NPTII; 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 NPTII 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.
Piñeiro
M
M
Laboratorio de Bioquímica y Biología Molecular, Universidad Politecnica Madrid, Spain.
García-Olmedo
F
F
Diaz
I
I
D002478:Cells, Cultured; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D066298:In Vitro Techniques; D019868:Kanamycin Kinase; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014026:Nicotiana; D014158:Transcription, Genetic / Q000187:drug effects
D012333:RNA, Messenger; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D003545:Cysteine; D004229:Dithiothreitol