Abstract
BackgroundThe amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples.ResultsThe DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions.ConclusionFlow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.
Highlights
The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development
Flow cytometry using DAPI DAPI is a DNA-binding fluorophore commonly used for fluorescence microscopy and flow cytometry [17,18,19,20]
We have assessed its utility for flow cytometric analysis of the DNA content of Arabidopsis chloroplasts that were isolated using a high salt procedure that avoids the use of DNase during the isolation of chloroplasts [11]
Summary
The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Chloroplasts arose from endosymbiosis between a cyanobacterium and a eukaryotic host and contain their own genome [1]. The chloroplast genome is a remnant of the original endosymbiont, a result of gene transfer to the nucleus over evolutionary time [2] and contains fewer than 100 protein-encoding genes in land plants [3]. The number of copies of the genome per chloroplast is not constant, but changes dramatically during development. The DNA is associated with RNA and proteins in complexes known as nucleoids [4].
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