Figure 3.
Analysis of JA, ethylene, and ROS as wound signals in DNRR.
(A) Upregulated genes in the JA biosynthesis pathway, as identified from RNA-seq data of Col-0 and coi1-2 leaf explants from t0 to 12 h after detachment. Numbers in each box are TPM values. P values were calculated by performing a paired t-test between TPM values of Col-0 and coi1-2. One replicate was regarded as one sample.
(B) Distribution of genes with scaled relative expression at each time point from RNA-seq data of Col-0 and coi1-2. Vertical lines indicate gene count density; horizontal lines indicate transcript levels at each time point. We independently scaled the expression levels at each time point for Col-0 and coi1-2. Arrow shows a group of genes highly upregulated in Col-0 but not in coi1-2 at 1 h after leaf detachment.
(C) GO analysis of genes highly upregulated in Col-0 but not in coi1-2 at 1 h after leaf detachment. Upregulation of these genes may be dependent on the COI1-mediated JA signaling pathway. See Supplemental Table 3 for the full list of GO terms.
(D) Analysis of promoter cis elements that may be targeted by the JA pathway. Genes listed in Supplemental Table 3 were used for cis element analysis. See Supplemental Table 3 for the full list of cis element analysis.
(E–G) Phenotype (E) and statistical (F and G) analysis of DNRR after ethylene or ROS inhibitor treatment. For ethylene treatment, Col-0 leaf explants were cultured on B5 medium with 4 μM of the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) for 6 or 24 h after detachment, then moved to ACC-free B5 medium for further culture. For the ROS inhibitor treatment, Col-0 leaf explants were cultured on B5 medium with 100 μM SHAM, 200 μM SHAM, or 1 mM GSH. Note that treatment with 200 μM SHAM could completely block rooting from leaf explants. Mock served as the control without chemical treatment. The rooting ratio (percentage of rooting leaves among all leaves tested at each time point) was tested (F and G). Error bars show the SEM of three biological replicates (n = 30 leaf explants per replicate), and individual values are indicated by dots (F and G).
(H) Map-based cloning showing mutations in the SUR1 gene. The mutation occurred in the first nucleotide of the fifth intron (G to A), resulting in a stop codon. Capital letters indicate exon nucleotides, and lowercase letters indicate intron nucleotides.
(I) Phenotype analysis of DNRR in wild-type Col-0 and sur1-21 after treatment with 200 μM SHAM. We tested more than 30 leaf explants of sur1-21, and all of them formed adventitious roots at 15 d after leaf detachment.
(J) Treatment with 0.1 μM of the synthetic auxin 1-naphtalene acetic acid (NAA) could partially rescue the rooting defect caused by 200 μM SHAM treatment at 15 d. Note that 82 out of 90 leaf explants formed adventitious roots upon co-treatment with SHAM and NAA.
Scale bars, 1 mm in (E, I, and J).