Immunogenic tagging of chloroplasts allows their isolation from defined cell types.
Chloroplast structure varies depending on cell type. Currently it is difficult to obtain insight into how differences in chloroplast structure relate to function, as it is often not possible to isolate chloroplasts from specific cells. To address this, we have developed an approach that involves labelling chloroplasts from individual cell types by placing the foreign yellow fluorescent protein (YFP) on their outer surface, and then isolating those labelled chloroplasts immunogenically. Cell specificity is achieved through the use of enhancer trap lines. When whole leaves are homogenized, a mixture of labelled and unlabelled chloroplasts is released, but magnetic beads coated with antibodies to the green fluorescent protein (GFP) allow the labelled chloroplasts to be isolated. Chloroplasts from spongy mesophyll, vascular and epidermal cells of Arabidopsis thaliana were obtained in this way, and semi-quantitative RT-PCR showed that the abundance of various chloroplast transcripts differed between these three cell types. The approach is based on genetic logic, and so could be applied to the isolation of various organelles or subcellular compartments from transformable organisms other than A. thaliana.
Truernit
Elisabeth
E
Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA, UK.
Hibberd
Julian M
JM
D001426:Bacterial Proteins / Q000235:genetics; D002458:Cell Fractionation; D002736:Chloroplasts / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure*; D017930:Genes, Reporter; D007125:Immunogenetics / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000166:cytology / Q000235:genetics* / Q000276:immunology; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria