Table S1

Primers used in this study"

Fig. 1

Relationship among ABCI8 orthologs in plants A: the phylogenetic tree of the sequence of BnABCI8 and its putative orthologs in different species. Numbers on branches are bootstrap values for 1000 replications. B: the amino acid sequences of BnABCI8 and putative orthologs in five species."

Fig. 2

Relative expression pattern of BnABCI8 genes Values are means ± SDs (n = 3)."

Fig. 3

Subcellular localization of BnABCI8 A, E: green fluorescence signals of transformed tobacco with the P35S::BnaA09.ABCI8:GFP and P35S::BnaC09.ABCI8:GFP constructs, respectively. B, F: red fluorescence of chloroplasts in tobacco. C, G: the image under bright field. D, H: the merged images from (A-C) and (E-G), respectively."

Fig. 4

Gene structure of BnABCI8 and plant identification of CRISPR-edited positive plants A: gene structure of BnA09.ABCI8 and BnC09.ABCI8; B: the electrophoretic identification of CRISPR-edited positive plants. The marker bands are 2 kb, 1 kb, 750 bp, 500 bp, and 200 bp, respectively, and the target band is 686 bp."

Fig. 5

Identification of T1 generation mutant A: the editing of some plants. The yellow mark indicates the PAM site and the inserted base, and the red dash indicates the deletion. B: the relative expression levels of BnABCI8 in mutants. * and ** mean significant differences at P < 0.05 and P < 0.01, respectively."

Fig. 6

Phenotypic observation of cotyledons in mutants A-C: 10-day-old wild type; D-F: 10-day-old single mutants; G-I: 10-day-old double mutants; J-L: 17-day-old double mutants."

Fig. 7

Phenotypic observation of mutants A: 56-day-old wild type; B: 56-day-old single mutants; C: 56-day-old double mutants."

Fig. 8

Phenotypic observation and chlorophyll content determination of true leaf in double mutant A, C: the comparison of true leaves and chlorophyll contents between double mutant and WT at 30 days old; B, D: the comparison of true leaves and chlorophyll contents between double mutant and WT at 56 days old. Values are presented as mean ± SD (n = 3). * and ** mean significant differences at P < 0.05 and P < 0.01, respectively."

Fig. 9

Relative expression levels of genes related to the chlorophyll synthesis pathway Value is mean ± SD (n = 3). * and ** mean significant differences at P < 0.05 and P < 0.01, respectively."

Fig. 10

Fe content in leaves a09 represents single mutants of the BnA09.ABCI8; a09 c09 represents double mutants of the BnA09.ABCI8 and BnC09.ABCI8. Value is mean ± SD (n = 3). * and ** mean significant differences at P < 0.05 and P < 0.01, respectively."

Fig. 11

Chloroplast ultrastructure of double mutant and WT A-C: the chloroplast structure of wild type; D-F: the chloroplast structure of double mutant; Cp: chloroplast; Tm: thylakoid membrane."