16S to 18S rDNA ratio; Arabidopsis; chloroplasts; cytokinin response factors; plastid copy numbers
The regulation of plastid density and size per cell by phytohormone-induced signaling cascades has been a focus of research many decades ago but has recently experienced a revival. Evidence for a connection between cytokinin levels, a transcription factor belonging to the cytokinin response factor group and the expression level of two plastid division proteins are likely just the beginning of a new field of endosymbiosis research that will require the screening of potential candidate genes under a variety of conditions in order to map the effects on chloroplast numbers per cell. We report here on a comparison of two methods for the determination of chloroplast copy numbers per cell. The direct counting of chloroplasts in 3-D models of cells reconstructed from optical sections using a fluorescence microscope equipped with an ApoTome is a highly accurate method but is time-consuming and tedious. In contrast, the determination of plastid to nuclear DNA ratio using the 16S and 18S rDNA genes, respectively, is a very rapid method suitable to screen large numbers of tissues, mutant seedlings or seedlings grown under different conditions. Although it targets the plastid DNA instead of the chloroplasts themselves, it correlates rather well with the counting method and can be recommended for initial investigations or for large-scale experimental set-ups.