Three dynamin-related protein 5B genes are related to plastid division in Physcomitrella patens

Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them (PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.