Two Groups of <i>Thellungiella salsuginea</i> RAVs Exhibit Distinct Responses and Sensitivity to Salt and ABA in Transgenic <i>Arabidopsis</i>

<div><p>Containing both AP2 domain and B3 domain, RAV (Related to ABI3/VP1) transcription factors are involved in diverse functions in higher plants. A total of eight <i>TsRAV</i> genes were isolated from the genome of <i>Thellungiella salsuginea</i> and could be divided into two groups (A- and B-group) based on their sequence similarity. The mRNA abundance of all <i>Thellungiella salsuginea TsRAVs</i> followed a gradual decline during seed germination. In <i>Thellungiella salsuginea</i> seedling, transcripts of <i>TsRAVs</i> in the group A (<i>A-TsRAVs</i>) were gradually and moderately reduced by salt treatment but rapidly and severely repressed by ABA treatment. In comparison, with a barely detectable constitutive expression, the transcriptional level of <i>TsRAVs</i> in the group B (<i>B-TsRAVs</i>) exhibited a moderate induction in cotyledons when confronted with ABA. We then produced the “gain-of-function” transgenic <i>Arabidopsis</i> plants for each <i>TsRAV</i> gene and found that only <i>35S</i>:<i>A-TsRAVs</i> showed weak growth retardation including reduced root elongation, suggesting their roles in negatively controlling plant growth. Under normal conditions, the germination process of all <i>TsRAVs</i> overexpressing transgenic seeds was inhibited with a stronger effect observed in <i>35S</i>:<i>A-TsRAVs</i> seeds than in <i>35S</i>:<i>B-TsRAVs</i> seeds. With the presence of NaCl, seed germination and seedling root elongation of all plants including wild type and <i>35S</i>:<i>TsRAVs</i> plants were retarded and a more severe inhibition occurred to the <i>35S</i>:<i>A-TsRAV</i> transgenic plants. ABA treatment only negatively affected the germination rates of <i>35S</i>:<i>A-TsRAV</i> transgenic seeds but not those of <i>35S</i>:<i>B-TsRAV</i> transgenic seeds. All <i>35S</i>:<i>TsRAVs</i> transgenic plants showed a similar degree of reduction in root growth compared with untreated seedlings in the presence of ABA. Furthermore, the cotyledon greening/expansion was more severely inhibited <i>35S</i>:<i>A-TsRAVs</i> than in <i>35S</i>:<i>B-TsRAVs</i> seedlings. Upon water deficiency, with a wider opening of stomata, <i>35S</i>:<i>A-TsRAVs</i> plants experienced a faster transpirational water loss than wild type and <i>35S</i>:<i>B-TsRAVs</i> lines. Taken together, our results suggest that two groups of TsRAVs perform distinct regulating roles during plant growth and abiotic defense including drought and salt, and A-TsRAVs are more likely than B-TsRAVs to act as negative regulators in the above-mentioned biological processes.</p></div

Sequence characterization of RAV family members of <i>Thellungiella salsuginea</i> and <i>Arabidopsis thaliana</i>.

<p>(A) Phylogenetic tree of the RAV family members in <i>Thellungiella salsuginea</i> and <i>Arabidopsis thaliana</i>. The phylogenetic tree was constructed using full-length protein sequences by the maximum-likelihood method with MEGA 5.0 and a bootstrap value of 1,000. The two major phylogenetic clades are designated as groups A and B. Shown on the right are diagrams of RAV proteins with information on the structure and position of different protein domains. (B) RAV subfamily-specific amino acids and their locations along the RAV full-length sequences. The amino acid sequences in boxes represent the conserved AP2 and B3 DNA-binding domains, which are characteristic of RAV transcription factors. The locations of the conserved YRG and RAYD elements are indicated as well. (C) Schematic illustrations of the types and distributions of motifs for each TsRAV family members with a same group. Motifs were identified using the MEME search tool and numerically marked according to their statistical significance (low <i>E</i>-value) in a descending order.</p

Cotyledon-greening analysis on <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seedlings.

<p>(A) Phenotypic comparison of wild-type and <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seedlings after grown on normal 1/2 MS medium (upper panel) or on 1/2 MS medium with 0.5 <i>μ</i>M ABA for 6 days (lower panel). (B) Cotyledon-greening percentages of <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seedlings after grown on 1/2 MS medium with 0.5 <i>μ</i>M ABA for 6 days. Each data bar represents the mean ± SE of three replicates. More than 100 seeds were measured in each replicate. Different letters indicate significant differences among means (<i>P</i><0.05 by Tukey’s test).</p

ABA sensitivity of <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> plants.

<p>(A) Germination rates of <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seeds on 1/2 MS media with 1 <i>μ</i>M ABA. Each data bar represents the means ± SE of three replicates. More than 100 seeds were measured in each replicate. (B) Inhibitory effect of 1 <i>μ</i>M ABA on <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seed germination rates. Each data bar represents the mean ± SE of three replicates. More than 50 seedlings were measured in each replicate. Different letters indicate significant differences among means (<i>P</i><0.05 by Tukey’s test). (C) Inhibitory effect of 30 <i>μ</i>M ABA on <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seedling root elongation. Seedlings were grown on normal media for 5 days before being transferred onto 1/2 MS medium with 30 <i>μ</i>M ABA and grown for other 6 days. Each data bar represents the mean ± SE of three replicates. More than 50 seedlings were measured in each replicate. Different letters indicate significant differences among means (<i>P</i><0.05 by Tukey’s test).</p

Expression of <i>TsRAV</i> genes during seed germination and in response to salt and ABA treatments.

<p>(A) qRT-PCR assay of <i>TsRAVs</i> transcription in <i>Thellungiella salsuginea</i> during seed germination. Analyses were performed on seeds at 0, 2, 4, and 6 DAI (days after imbibition). The transcription levels of genes were quantified relative to that of <i>actin</i>. Each data bar represents the means ± SE (Standard Error) of three replicates. (B) Semi-quantitative RT-PCR assay of <i>TsRAVs</i> transcription in <i>Thellungiella salsuginea</i> seedlings upon 200 mM NaCl treatment. Total RNA was extracted at various time intervals from leaves and roots of 15-d-old seedlings. The cDNA samples are normalized using an <i>actin</i> gene as an internal control. (C) Semi-quantitative RT-PCR assay of <i>TsRAVs</i> transcription in <i>Thellungiella salsuginea</i> seedlings upon 5 <i>μ</i>M ABA treatment. Total RNA was extracted at various time intervals from leaves and roots of 15-d-old seedlings. The cDNA samples are normalized using an <i>actin</i> gene as an internal control.</p

Phenotypic characterization of <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> plants under normal conditions.

<p>(A) Primary root length of 7-d-old <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seedlings grown on 1/2 MS media. Each data bar represents the means ± SE of three replicates. More than 50 seedlings were measured for each replicates. Different letters indicate significant differences among means (<i>P</i><0.05 by Tukey’s test). (B) Germination rates of <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> seeds during a 5-day period on normal 1/2 MS media. Each data bar represents the means ± SE of three replicates. More than 100 seeds were measured in each replicated.</p

Different water loss rates of wild-type and <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> plants.

<p>(A) Weight loss in fresh leaves under water deficit of 4-week-old wild-type and <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> plants. Each data bar represents the mean ± SE of three replicates. (B) Width/length ratio of the rosette leaf stomatal aperture of 4-week-old wild-type and <i>35S</i>:<i>TsRAVs</i> transgenic <i>Arabidopsis</i> plants. Each data bar represents the mean ± SE of three replicates and different letters indicate significant differences among means (<i>P</i><0.05 by Tukey’s test).</p

Migration and Transformation Characteristics of Niclosamide in a Soil–Plant System

The molluscicide niclosamide is found in most of the wetlands of China. The migration and transformation pathways, and degradation kinetics of niclosamide in the plant–soil system was analyzed by with the use of potting experiment. Experimental results showed that degradation of niclosamide in rhizosphere soil fit the first-order kinetics, and microorganisms played an important role in the degradation of niclosamide. It was found that niclosamide degrades to form a series of aromatic intermediate products both in soil and plants. Niclosamide could be absorbed from soil to plant by the root and then migrate to the stem. At an initial concentration of niclosamide of 2.11 mg·kg^–1 in soil, the maximum residue of niclosamide in <i>Artemisia somai</i> aerial was 2.47 mg·kg^–1 after 10 days of cultivation. This value is close to the pollution maximum residue limit (3 mg·kg^–1) in rice, and niclosamide and its intermediates can remain about 43 days in plants. The experimental results demonstrate that the use of niclosamide in wetlands would have some risk in edible plants and was harmful for human health