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

Abstract

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 TsRAV genes were isolated from the genome of Thellungiella salsuginea and could be divided into two groups (A- and B-group) based on their sequence similarity. The mRNA abundance of all Thellungiella salsuginea TsRAVs followed a gradual decline during seed germination. In Thellungiella salsuginea seedling, transcripts of TsRAVs in the group A (A-TsRAVs) 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 TsRAVs in the group B (B-TsRAVs) exhibited a moderate induction in cotyledons when confronted with ABA. We then produced the "gain-of-function" transgenic Arabidopsis plants for each TsRAV gene and found that only 35S:A-TsRAVs showed weak growth retardation including reduced root elongation, suggesting their roles in negatively controlling plant growth. Under normal conditions, the germination process of all TsRAVs overexpressing transgenic seeds was inhibited with a stronger effect observed in 35S:A-TsRAVs seeds than in 35S:B-TsRAVs seeds. With the presence of NaCl, seed germination and seedling root elongation of all plants including wild type and 35S:TsRAVs plants were retarded and a more severe inhibition occurred to the 35S:A-TsRAV transgenic plants. ABA treatment only negatively affected the germination rates of 35S:A-TsRAV transgenic seeds but not those of 35S:B-TsRAV transgenic seeds. All 35S:TsRAVs 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 35S:A-TsRAVs than in 35S:B-TsRAVs seedlings. Upon water deficiency, with a wider opening of stomata, 35S:A-TsRAVs plants experienced a faster transpirational water loss than wild type and 35S:B-TsRAVs 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.

Fig 1. Sequence characterization of RAV family members of Thellungiella salsuginea and Arabidopsis thaliana.

(A) Phylogenetic tree of the RAV family members in Thellungiella salsuginea and Arabidopsis thaliana. 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 E-value) in a descending order.

Fig 2. Expression of TsRAV genes during seed germination and in response to salt and ABA treatments.

(A) qRT-PCR assay of TsRAVs transcription in Thellungiella salsuginea 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 actin. Each data bar represents the means ± SE (Standard Error) of three replicates. (B) Semi-quantitative RT-PCR assay of TsRAVs transcription in Thellungiella salsuginea 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 actin gene as an internal control. (C) Semi-quantitative RT-PCR assay of TsRAVs transcription in Thellungiella salsuginea seedlings upon 5 μM 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 actin gene as an internal control.

Fig 3. Phenotypic characterization of 35S:TsRAVs transgenic Arabidopsis plants under normal conditions.

(A) Primary root length of 7-d-old 35S:TsRAVs transgenic Arabidopsis 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 (P<0.05 by Tukey’s test). (B) Germination rates of 35S:TsRAVs transgenic Arabidopsis 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.

Fig 4. NaCl sensitivity of 35S:TsRAVs transgenic Arabidopsis plants.

(A) Germination rates of 35S:TsRAVs transgenic Arabidopsis seeds on 1/2 MS media with 100 mM NaCl. Each data bar represents the means ± SE of three replicates. More than 100 seeds were measured in each replicate. (B) Inhibitory effect of 100 mM NaCl on 35S:TsRAVs transgenic Arabidopsis 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 (P<0.05 by Tukey’s test). (C) Inhibitory effect of 200 mM NaCl on 35S:TsRAVs transgenic Arabidopsis seedling root elongation. Seedlings were grown on normal media for 5 days before being transferred onto 1/2 MS medium with 200 mM NaCl 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 (P<0.05 by Tukey’s test)

Fig 5. ABA sensitivity of 35S:TsRAVs transgenic Arabidopsis plants.

(A) Germination rates of 35S:TsRAVs transgenic Arabidopsis seeds on 1/2 MS media with 1 μM 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 μM ABA on 35S:TsRAVs transgenic Arabidopsis 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 (P<0.05 by Tukey’s test). (C) Inhibitory effect of 30 μM ABA on 35S:TsRAVs transgenic Arabidopsis seedling root elongation. Seedlings were grown on normal media for 5 days before being transferred onto 1/2 MS medium with 30 μM 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 (P<0.05 by Tukey’s test).

Fig 6. Cotyledon-greening analysis on 35S:TsRAVs transgenic Arabidopsis seedlings.

(A) Phenotypic comparison of wild-type and 35S:TsRAVs transgenic Arabidopsis seedlings after grown on normal 1/2 MS medium (upper panel) or on 1/2 MS medium with 0.5 μM ABA for 6 days (lower panel). (B) Cotyledon-greening percentages of 35S:TsRAVs transgenic Arabidopsis seedlings after grown on 1/2 MS medium with 0.5 μM 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 (P<0.05 by Tukey’s test).

Fig 7. Different water loss rates of wild-type and 35S:TsRAVs transgenic Arabidopsis plants.

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