Expression of the plastid-located glutamine synthetase of Medicago truncatula. Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids.
In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2). A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids. Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs. Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ. Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2). This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules. Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive. Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope. Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules. These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.
Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
Paula M
PM
Melo
Lígia M
LM
Lima
Isabel M
IM
Santos
Helena G
HG
Carvalho
Julie V
JV
Cullimore
D000595:Amino Acid Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D029902:Medicago / Q000201:enzymology / Q000235:genetics* / Q000382:microbiology; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology* / Q000382:microbiology / Q000648:ultrastructure; D018087:Plastids / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013559:Symbiosis / Q000235:genetics
D018076:DNA, Complementary; D004792:Enzyme Precursors; D010940:Plant Proteins; D005974:Glutamate-Ammonia Ligase