Molecular Cloning, Characterization and Expression Analysis of the <i>SAMS</i> Gene during Adventitious Root Development in IBA-Induced Tetraploid Black Locust

<div><p>S-Adenosylmethionine synthetase (SAMS) catalyzes the synthesis of S-adenosylmethionine (SAM), a precursor for ethylene and polyamine biosynthesis. Here, we report the isolation of the 1498 bp full-length cDNA sequence encoding tetraploid black locust (<i>Robinia pseudoacacia</i> L.) SAMS (<i>TrbSAMS</i>), which contains an open reading frame of 1179 bp encoding 392 amino acids. The amino acid sequence of TrbSAMS has more than 94% sequence identity to SAMSs from other plants, with a closer phylogenetic relationship to SAMSs from legumes than to SAMS from other plants. The TrbSAMS monomer consists of N-terminal, central, and C-terminal domains. Subcellular localization analysis revealed that the TrbSAMS protein localizes mainly to in the cell membrane and cytoplasm of onion epidermal cells and <i>Arabidopsis</i> mesophyll cell protoplasts. Indole-3-butyric acid (IBA)-treated cuttings showed higher levels of <i>TrbSAMS</i> transcript than untreated control cuttings during root primordium and adventitious root formation. <i>TrbSAMS</i> and its downstream genes showed differential expression in shoots, leaves, bark, and roots, with the highest expression observed in bark. IBA-treated cuttings also showed higher SAMS activity than control cuttings during root primordium and adventitious root formation. These results indicate that <i>TrbSAMS</i> might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings.</p></div

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<p><b>Activities of nitrate reductase (NR, μM NO3 g^–1FW h^-1) (A and B) and nitrite reductase (NiR, mmol NO2- h^-1 mg^-1protein) (C and D) in roots and leaves of <i>P. simonii</i> as affected by salt treatment and nitrogen nutrition.</b> White boxes indicate ammonium only; grey boxes indicate ammonium with salt; black boxes indicate nitrate only, and striped boxes indicate nitrate with salt. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant difference. N, N nutrition; S, salt stress; N*S, interaction of N nutrition and salt treatment. *P< 0.05; **P <0.01; *** P <0.001; ns, not significant.</p

Amino acid sequence alignment of TrbSAMS with SAMS sequences from other plant species.

<p>GenBank accession numbers for nucleotide sequences: <i>Glycine max</i>, XP_003550837.1; <i>Cajanus cajan</i>, AEY85025.1; <i>Glycine soja</i>, ABY25855.1; <i>Ricinus communis</i>, XP_002512570.1; <i>Medicago truncatula</i>, XP_003609861.1; <i>Vitis vinifera</i>, XP_002266358.1; <i>Populus trichocarpa</i>, XP_002312296.1; <i>Cucumis sativus</i>, XP_004168041.1; <i>Prunus persica</i>, 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.</p

Anatomical changes in tetraploid black locust softwood cuttings undergoing IBA-induced adventitious root development.

<p>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.</p

Nitrate and Ammonium Contribute to the Distinct Nitrogen Metabolism of <i>Populus simonii</i> during Moderate Salt Stress - Fig 6

<p><b>Amino compounds in roots (A) and leaves (B) of <i>P. simonii</i> as affected by salt treatment and nitrogen nutrition.</b> Amino acids are grouped together deriving from the same pathway. Presented are the percent distributions of these biosynthetic groups, and mean values of sums of amino compounds of each group of three plants and SE. Black: aspartate, threonine, isoleucine, methionine, lysine; light grey: serine, glycine, cysteine; white: glutamate, histidine, arginine, proline; striped: phenylalanine, tyrosine; dark grey: alanine, leucine, valine.</p

Phylogenetic tree of TrbSAMS and other plant SAMS proteins.

<p>GenBank accession number for nucleotides sequences: <i>Glycine max</i>, XP_003550837.1; <i>Cajanus cajan</i>, AEY85025.1; <i>Glycine soja,</i> ABY25855.1; <i>Ricinus communis</i>, XP_002512570.1; <i>Medicago truncatula</i>, XP_003609861.1; <i>Vitis vinifera</i>, XP_002266358.1; <i>Populus trichocarpa</i>, XP_002312296.1; <i>Cucumis sativus</i>, XP_004168041.1; <i>Theobroma cacao</i>, EOY06891.1; <i>Gossypium hirsutum</i>, ADN96174.1; <i>Prunus persica</i> AGF95108.1; <i>Lycoris radiata</i> AFC88125.1; <i>Arabidopsis thaliana</i>, NP_188365.1; <i>Oryza officinalis</i> CAJ45561.1; <i>Gladiolus grandiflorus</i>, 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.</p

Predicated tertiary structure of TrbSAMS protein established by SWISS-MODE.

<p>The amino and carboxylic termini are labeled N and C, respectively. The entrances of substrate binding sites are marked with white arrows.</p

Relative expression levels of <i>TrbSAMS</i> (A), <i>TrbSAMDC</i> (B), <i>TrbPAO</i> (C), and <i>TrbACS</i> (D) during different IBA-induced and untreated rooting phases in softwood cuttings of tetraploid black locust.

<p>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.</p

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.

<p>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.</p

Morphological changes in tetraploid black locust cuttings undergoing adventitious root development in a sand bed.

<p>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.</p