In vivo effects of NbSiR silencing on chloroplast development in Nicotiana benthamiana.
Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.
Kang
Yong-Won
YW
Department of Biology, Yonsei University, Seoul, 120-749, Korea.
Lee
Jae-Yong
JY
Jeon
Young
Y
Cheong
Gang-Won
GW
Kim
Moonil
M
Pai
Hyun-Sook
HS
D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003545:Cysteine / Q000494:pharmacology; D018744:DNA, Plant / Q000378:metabolism; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000032:analysis; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics* / Q000502:physiology; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013025:Glycine max / Q000235:genetics; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure
D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; D003545:Cysteine