Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants.
Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.
Kim
Yu-Kyung
YK
Laboratory of Plant Genomics, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea.
Lee
Jae-Yong
JY
Cho
Hye Sun
HS
Lee
Sang Sook
SS
Ha
Hyun Jung
HJ
Kim
Sunghoon
S
Choi
Doil
D
Pai
Hyun-Sook
HS
D017360:Arabidopsis / Q000201:enzymology; D002736:Chloroplasts / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D005975:Glutamate-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D015388:Organelles; D010641:Phenotype; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000378:metabolism / Q000821:virology; D010942:Plant Viruses / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012714:Serine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020524:Thylakoids; D014026:Nicotiana / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012714:Serine-tRNA Ligase; D005975:Glutamate-tRNA Ligase