Table 1.
Primers used in the study.
Figure 1.
Morphological changes in tetraploid black locust cuttings undergoing adventitious root development in a sand bed.
a. Softwood cuttings before cutting. b. A white callus appeared at 10 days after cutting. c. A yellow callus appeared and tiny adventitious roots emerged (root primordium) at 15 days after cutting. d. Adventitious root formation and elongation at 20 days after cutting.
Figure 2.
Anatomical changes in tetraploid black locust softwood cuttings undergoing IBA-induced adventitious root development.
a. Cross-section of the stem before cutting. b. Parenchyma cells of callus at 10 days after cutting. c. Root primordium from the parenchyma cells at the junction between the pith rays and cortex at 15 days after cutting. d. Adventitious roots appearing at 20 days after cutting. Scale bars, 20 µm.
Figure 3.
Amino acid sequence alignment of TrbSAMS with SAMS sequences from other plant species.
GenBank accession numbers for nucleotide sequences: Glycine max, XP_003550837.1; Cajanus cajan, AEY85025.1; Glycine soja, ABY25855.1; Ricinus communis, XP_002512570.1; Medicago truncatula, XP_003609861.1; Vitis vinifera, XP_002266358.1; Populus trichocarpa, XP_002312296.1; Cucumis sativus, XP_004168041.1; Prunus persica, AGF95108.1. The same and similar amino acid residues are highlighted in black and gray respectively; the underlined sequences indicate NADP-binding sites and substrate specificity domains. Three conserved motifs are indicated in red. Two SAM synthetase signature motifs are underlined.
Figure 4.
Phylogenetic tree of TrbSAMS and other plant SAMS proteins.
GenBank accession number for nucleotides sequences: Glycine max, XP_003550837.1; Cajanus cajan, AEY85025.1; Glycine soja, ABY25855.1; Ricinus communis, XP_002512570.1; Medicago truncatula, XP_003609861.1; Vitis vinifera, XP_002266358.1; Populus trichocarpa, XP_002312296.1; Cucumis sativus, XP_004168041.1; Theobroma cacao, EOY06891.1; Gossypium hirsutum, ADN96174.1; Prunus persica AGF95108.1; Lycoris radiata AFC88125.1; Arabidopsis thaliana, NP_188365.1; Oryza officinalis CAJ45561.1; Gladiolus grandiflorus, ADM18304.1. Underlining indicates the amino acid sequence of TrbSAMS cloned in this study. The phylogenetic tree was constructed using the program MEGA 5.0. The numbers shown at internal nodes indicate the occurrence of these nodes in 1000 replicates, and the bar represents 20% sequence divergence.
Figure 5.
Predicated tertiary structure of TrbSAMS protein established by SWISS-MODE.
The amino and carboxylic termini are labeled N and C, respectively. The entrances of substrate binding sites are marked with white arrows.
Figure 6.
Subcellular localization of the TrbSAMS-GFP fusion protein.
(A) Subcellular localization of the TrbSAMS-GFP fusion protein in onion epidermal cells. GFP fluorescence (GFP; green pseudocolor), optical photomicrographs (bright field) and an overlay of bright and GFP fluorescence illumination (merged) are shown; the arrows point to the nucleus of the cells. (B) Subcellular localization of the TrbSAMS-GFP fusion protein in Arabidopsis protoplasts. Images are GFP fluorescence (GFP; green pseudocolor), chlorophyll fluorescence (chlorophyll; red pseudocolor), optical photomicrographs (bright field) and merged (optical photomicrographs, chlorophyll fluorescence and GFP fluorescence). Data shown are representative of three independent experiments (n = 3). Scale bars, 200 µm.
Figure 7.
Relative expression levels of TrbSAMS (A), TrbSAMDC (B), TrbPAO (C), and TrbACS (D) during different IBA-induced and untreated rooting phases in softwood cuttings of tetraploid black locust.
Initiation phase (I). Callus induction phase (C). Root primordia formation phase (RP). Adventitious root formation and elongation phase (AR). Error bars represent the standard deviation (SD) calculated from three biological replicates with IBA, CK (n = 3). CK, control treatment; IBA, indole-3-butanoic acid.
Figure 8.
Expression of TrbSAMS, TrbSAMDC, TrbPAO, and TrbACS in the shoot, leaf, bark, and root.
Error bars represent the standard deviation (SD) calculated from three biological replicates (n = 3).
Figure 9.
SAMS activity (A), polymines contents (B), ACS activity (C), and ethylene production (D) during the different IBA-induced and untreated rooting phase in softwood cuttings of tetraploid black locust.
Initiation phase (I), callus induction phase (C), root primordia formation phase (RP), adventitious root formation and elongation phase (AR). Error bars represent the standard deviation (SD) calculated from three biological replicates with IBA, CK (n = 3). CK, control treatment; IBA, indole-3-butanoic acid.