Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur.
Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5' regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial beta-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of beta-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cell cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0-24 h) cultivation periods compared with cells grown in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.
Takahashi
H
H
Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology, Chiba University, Japan.
Saito
K
K
D001483:Base Sequence; D002478:Cells, Cultured; D002736:Chloroplasts / Q000201:enzymology; D003547:Cysteine Synthase / Q000096:biosynthesis*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000096:biosynthesis; D007527:Isoenzymes / Q000096:biosynthesis; D009584:Nitrogen / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018724:Spinacia oleracea / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology; D013455:Sulfur / Q000494:pharmacology*; D014158:Transcription, Genetic
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013455:Sulfur; D003547:Cysteine Synthase; D005966:Glucuronidase; D009584:Nitrogen