Additional file 1 of OsNAC103, a NAC Transcription Factor, Positively Regulates Leaf Senescence and Plant Architecture in Rice

Abstract

Additional file 1. Fig. S1: Phylogenic analysis and sequence analysis of OsNAC103. A An unrooted phylogenetic tree of stress-responsive NAC (SNAC) proteins in rice and Arabidopsis. SNAC-A and -B are two subgroups of SNAC proteins. The tree was drawn using the Neighbor-Joining method in the MEGA 11.0 program. B Multiple sequence alignments between OsNAC103 and other members of the NAC subfamily in rice. (a)–(e) represent five highly conservative regions. Fig. S2: Phenotype and overexpression levels of OsNAC103-OE lines in T0 generation. A Phenotypes of OsNAC103-OE lines. B Overexpression levels of OsNAC103-OE lines using qRT-PCR. OE1, OE2, OE6, OE7 and OE9 are five independent OsNAC103-OE lines. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S3: Sequencing analysis of osnac103 mutants (CR2 and CR5) by CRISPR-Cas9 system. A Target sites of CRISPR-Cas9 for OsNAC103. Solid boxes, exons; hollow box, 5′-UTR; hollow pentagon, 3′-UTR; the lines, introns; Target1, Target2 and Target3 represent three targets of OsNAC103, respectively. B–G Mutation sites of CR2 and CR5. Red boxes mean the position of mutations.—means deletion. Red underlines mean the position of PAM. Fig. S4: OsNAC103 positively regulates leaf senescence in rice. A–C Phenotype of OsNAC103-OE lines and osnac103 mutants during the vegetative growth stage. Bars = 20 cm. B indicates the magnified figure in A. (C) Phenotype of OsNAC103-OE lines and osnac103 mutants in the field. D, E Phenotype and total chlorophyll contents of different leaves in OsNAC103-OE lines and osnac103 mutants. Leaf-2, Leaf-3, Leaf-4, Leaf-5 represent the second, third, fourth and fifth leaves of the rice plant from top down, respectively. Bar = 5 cm. OE2 and OE7 are two independent OsNAC103-OE lines. CR2 and CR5 are two allelic mutants. Values are shown as means ± SD, n = 3. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S5: Agronomic traits of OsNAC103-OE lines and osnac103 mutants. A Panicle length, number of primary branches and 100 grains weight of OsNAC103-OE lines and osnac103 mutants. Values are shown as means ± SD, n ≥ 10 individual plants. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S6: Expression analysis of genes associated with tiller angle and shoot gravitropism in OsNAC103-OE lines and osnac103 mutants. RNAs were extracted from penultimate leaves of WT, OE lines and osnac103 mutants at vegetative stage, and qRT-PCR was performed to analyze the relative expression levels of different genes. OE2 and OE7 are two independent OsNAC103-OE lines. CR2 and CR5 are two allelic mutants. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S7: Interaction of OsNAC103 and the promoters of leaf senescence-associated genes. Yeast cells transformed with the indicated plasmids were grown on selective SD/-Ura/-Leu medium added with X-Gal. The interaction of pLacZi-proOsMADS14 and OsFTL12 effector was as a positive control. Fig. S8: Alternative splices of OsNAC103 and the blast of their amino acid sequences. A Three alternative splicing forms of OsNAC103 indicated as Loc_Os07g48450.1, Loc_Os07g48450.2 and Loc_Os07g48450.3, respectively. Solid boxes, exons; hollow box, 5′-UTR; hollow pentagon, 3′-UTR; the lines, introns. B Alignment of amino acids sequence for the Loc_Os07g48450.1, Loc_Os07g48450.2 and Loc_Os07g48450.3. C RT-PCR analysis of different transcripts of Loc_Os07g48450. cDNA was obtained from leaves at ripening stage by the reverse transcription reaction. RT-PCR were performed to analyze the possible transcripts (indicated by red arrows). F, R and R’ were different primers indicated in (A). M1 and M2 were DNA ladder