Fig. 1: A point mutation in ATH rescues the developmental defects of atrnh1c and weakens the binding of template DNA in replication.

a Photographs and chlorophyll fluorescence images of 21-day-old Col-0, atrnh1c, acs1, acs1^ATH::ATH, and acs1^ATH::ATH-GFP plants. Experiments were repeated three times with similar results. Chlorophyll Fv/Fm values are presented in the lower panel. Scale bar, 1 cm. b Comparison of chloroplasts in protoplasts from the leaves of 21-day-old Col-0, atrnh1c, acs1, acs1^ATH::ATH, and acs1^ATH::ATH-GFP plants. Chloroplasts are distinguished by chlorophyll autofluorescence (red). Scale bars, 5 μm. c Schematic representations of the ATH gene and its encoded protein. Exons are in black and 5′- and 3′-UTRs are in white. The positions of the point mutation and amino acid substitution are indicated. d Multiple sequence alignment between Twinkles in different plant species. The CXRXKC elements and R166 in ATH are shown. Proteins are designated with abbreviations as follows: Atr, Amborella trichopoda; Sm, Selaginella moellendorffii; Pp, Physcomitrium patens; Mp, Marchantia polymorpha; Os, Oryza sativa; Sb, Sorghum bicolor; Zm, Zea mays; Nn, Nelumbo nucifera; Sl, Solanum lycopersicum; Ls, Lactuca sativa; Cs, Cucumis sativus; At, Arabidopsis thaliana; Bn, Brassica napus; Ppe, Prunus persica; Rc, Rosa chinensis; Jr, Juglans regia; Gm, Glycine max; Mt, Medicago truncatula. e The single-strand DNA binding activities of ATH and ATH(R166K) were evaluated by EMSA. Ten-hundred-fold unlabeled probes were used for competition. Equal amounts of the two proteins were used for the EMSA assays. Experiments were repeated three times with similar results. f ChIP-qPCR analysis showed the enrichment of ATH-GFP and ATH(R166K)-GFP at cp-rDNA regions. Col-0 was used as the negative control. Three biological replicates were performed. The graphs represent the mean ± SD. **P < 0.01; ***P < 0.001 by unpaired two-sided t test. Source data are provided as a Source Data file.