Table 1.
RBOH genes identified in this study.
Subcellular localization predications indicated that the ROBHs were all located in the cell membrane.
Fig 1.
Phylogenetic and comparative analysis of RBOH genes from various plants.
(A) A summary of the species phylogeny and the number of RBOH genes in each species. (B) Phylogenetic analysis of RBOH proteins from six Rosaceae species and Arabidopsis.
Fig 2.
Chromosomal distribution and duplication of RBOH genes.
(A) Chromosomal distribution of the RBOH genes among six Rosaceae species. (B) Synteny analysis of the apple RBOH genes. Syntenic apple RBOH genes are linked by lines.
Fig 3.
Phylogenetic relationships, motif composition, conserved domains, and gene structures of RBOH proteins and their corresponding genes from six Rosaceae species.
(A) The phylogenetic tree was drawn based on the full-length protein sequences of RBOHs using MEGA 7.0. (B) Motifs in RBOH proteins. Ten motifs are indicated by different colored boxes. (C) Conserved domains of the RBOH proteins. Six conserved domains are indicated with different colored boxes. (D) Gene structures of the RBOHs. Introns, exons and untranslated regions (UTRs) are indicated by gray lines, yellow rectangles and green rectangles, respectively.
Fig 4.
Effects of DPI on adventitious root formation of apple stem cuttings.
(A) Adventitious root formation in apple stem cuttings after 18 days in rooting medium (control) or rooting medium supplemented with DPI (5 μM, 10 μM, and 20 μM). Percent rooting (B) and adventitious root number per cutting (C) were counted at 18 days. Bars indicate mean and Standard Deviation (SD) of three biological replicates; n = 6 individuals per replicate.
Fig 5.
Effects of exogenous H2O2 on adventitious root formation of apple stem cuttings.
(A) Adventitious root formation in apple stem cuttings after 18 days in rooting medium or rooting medium supplemented with H2O2 (1 mM, 5 mM, and 25 mM). “+” represents presence, “−” represents absence. The concentrations of IBA and NAA were 0.5 mg L−1 and 0.1 mg L−1, respectively. (B) Percent rooting. Bars indicate mean and SE from three biological replicates; n = 6 individuals per replicate. Asterisks indicate significant differences between stem cuttings subcultured in 1/2 MS and stem cuttings subcultured in 1/2 MS + IBA +NAA (Student’s t-test, **P < 0.01, *P < 0.05). (C) Adventitious root number per cutting. Adventitious root number per cutting was counted at 18 days. Bars indicate mean and SE from three biological replicates; n = 6 individuals per replicate. The statistical analysis was conducted using Duncan’s multiple range test (P < 0.05). The different lowercase letters indicate significant differences.
Fig 6.
Reactive oxygen species are participated in adventitious root formation in apple stem cuttings.
Stem cross-sections of ‘Gala’ apples taken at 3, 6, 9, and 12 days after subculture on rooting medium and stained with toluidine blue (A-D) and Nitro blue tetrazolium (NBT; O2-) (E-H), respectively. Scale bars (1000 μm, 500 μm, and 200 μm) are indicated in the figures. Pictures in red boxes show a 20⊆ view of the stem cuttings in (F, G). The rectangle indicates NBT-stained root tip in (H).
Fig 7.
Relative expression levels of MdRBOH genes during the adventitious rooting process.
Relative transcript levels of 10 MdRBOH genes were measured in stem tissue after the cuttings had been transferred to rooting medium for 0, 3, 6, or 9 days. Transcript levels were measured by qRT-PCR using apple EF1α as the reference gene. Data are means ± SD of three biological replicates. Refer to S1 Table for primers. The statistical analysis was conducted using Duncan’s multiple range test (P < 0.05). The different lowercase letters indicate significant differences.