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

Yang S, Luo C, Song Y, Wang J - PLoS ONE (2016)

Bottom Line: 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.All 35S:TsRAVs transgenic plants showed a similar degree of reduction in root growth compared with untreated seedlings in the presence of ABA.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.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin, 300072, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0153517.g001: 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.

Mentions: The Arabidopsis genome contains six RAVs that contain both AP2 domain and B3 domain [30] and AtRAVs share 35–79% amino acid identity throughout their full-length sequences. Using RAV sequences from Arabidopsis and other species as query sequences to search the T. salsuginea genome database, a total of 8 TsRAVs were isolated with the predicted full-length proteins ranging from 320 to 384 amino acids (S2 Table). In the phylogenetic tree, eight TsRAVs and six AtRAVs obviously split into two groups (Fig 1A). The group A includes TsRAV1-4 and AtRAV1 (At1g13260), AtRAV2/TEM2 (AT1g68840), AtRAV3/RAV1L (At3g25730) and AtEDF1/TEM1 (AT1g25560), and the group B includes TsRAV5-8 and two AtRAVs (At1g51120 and At1g50680) (Fig 1A). At their N-terminal regions, all eight TsRAVs contain an AP2 DNA-binding domain that recognizes a consensus CAACA sequence [31] and includes the conserved YRG and RAYD elements as well as a conserved 7-aa WAAEIRD box motif [32] (Fig 1A). At the C-terminal regions, all eight TsRAVs contain a B3 domain that recognizes a consensus CACCTG sequence and a 15-aa B3 repression domain (BRD) (GNSKTLRLFGVNMEC) that is responsible for the repressive activity in many B3 super-family members (Fig 1A). A short peptide composed of five amino acid residues, R/KLFGV, has been demonstrated to be crucial to maintain the repressive activity of the BRD domain [33]. In this work, TsRAVs in group A and group B include a RLFGV and M/KLFGV core sequence, respectively, which was also identified in rice RAV homologues [33]. The function of RAVs as transcriptional repressors has been demonstrated in several plant species. For example, TEM1 and GmRAV can repress flowering by binding to and repressing the promoters of FT genes in Arabidopsis and soybean, respectively [34,35]. Notably, each A-TsRAV but not a single B-TsRAV contains a nuclear localization sequence, which might contribute significantly to their roles as transcriptional regulators (Fig 1A). In addition to the BRD domain, a close comparison of the amino acid sequences between A-TsRAVs and B-TsRAVs revealed that they are characterized by distinctly different amino acids at some highly conserved positions and these group-specific residues might in turn affect the secondary structure of RAV proteins as well as their DNA-binding activities (Fig 1B). We also analyzed the motif composition of AtRAVs and TsRAVs using the MEME program. In total, 17 conserved motifs were detected and some of them appeared to be highly divergent between two groups (Fig 1C). For example, motifs 5 and 12 located in front of the AP2 domain, motif 9 within the B3 domain and motif 8 at the C-end of the RAV protein are highly conserved in the group A-TsRAVs, but are absent in the B-group RAVs. On the contrary, motif 10 within the AP2 domain, motifs 13 and 16 within the B3 domain and motif 14 located in front of the BRD domain are present in all group B-TsRAVs but missing in the group A-TsRAVs (Fig 1C).


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

Yang S, Luo C, Song Y, Wang J - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836749&req=5

pone.0153517.g001: 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.
Mentions: The Arabidopsis genome contains six RAVs that contain both AP2 domain and B3 domain [30] and AtRAVs share 35–79% amino acid identity throughout their full-length sequences. Using RAV sequences from Arabidopsis and other species as query sequences to search the T. salsuginea genome database, a total of 8 TsRAVs were isolated with the predicted full-length proteins ranging from 320 to 384 amino acids (S2 Table). In the phylogenetic tree, eight TsRAVs and six AtRAVs obviously split into two groups (Fig 1A). The group A includes TsRAV1-4 and AtRAV1 (At1g13260), AtRAV2/TEM2 (AT1g68840), AtRAV3/RAV1L (At3g25730) and AtEDF1/TEM1 (AT1g25560), and the group B includes TsRAV5-8 and two AtRAVs (At1g51120 and At1g50680) (Fig 1A). At their N-terminal regions, all eight TsRAVs contain an AP2 DNA-binding domain that recognizes a consensus CAACA sequence [31] and includes the conserved YRG and RAYD elements as well as a conserved 7-aa WAAEIRD box motif [32] (Fig 1A). At the C-terminal regions, all eight TsRAVs contain a B3 domain that recognizes a consensus CACCTG sequence and a 15-aa B3 repression domain (BRD) (GNSKTLRLFGVNMEC) that is responsible for the repressive activity in many B3 super-family members (Fig 1A). A short peptide composed of five amino acid residues, R/KLFGV, has been demonstrated to be crucial to maintain the repressive activity of the BRD domain [33]. In this work, TsRAVs in group A and group B include a RLFGV and M/KLFGV core sequence, respectively, which was also identified in rice RAV homologues [33]. The function of RAVs as transcriptional repressors has been demonstrated in several plant species. For example, TEM1 and GmRAV can repress flowering by binding to and repressing the promoters of FT genes in Arabidopsis and soybean, respectively [34,35]. Notably, each A-TsRAV but not a single B-TsRAV contains a nuclear localization sequence, which might contribute significantly to their roles as transcriptional regulators (Fig 1A). In addition to the BRD domain, a close comparison of the amino acid sequences between A-TsRAVs and B-TsRAVs revealed that they are characterized by distinctly different amino acids at some highly conserved positions and these group-specific residues might in turn affect the secondary structure of RAV proteins as well as their DNA-binding activities (Fig 1B). We also analyzed the motif composition of AtRAVs and TsRAVs using the MEME program. In total, 17 conserved motifs were detected and some of them appeared to be highly divergent between two groups (Fig 1C). For example, motifs 5 and 12 located in front of the AP2 domain, motif 9 within the B3 domain and motif 8 at the C-end of the RAV protein are highly conserved in the group A-TsRAVs, but are absent in the B-group RAVs. On the contrary, motif 10 within the AP2 domain, motifs 13 and 16 within the B3 domain and motif 14 located in front of the BRD domain are present in all group B-TsRAVs but missing in the group A-TsRAVs (Fig 1C).

Bottom Line: 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.All 35S:TsRAVs transgenic plants showed a similar degree of reduction in root growth compared with untreated seedlings in the presence of ABA.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.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Science and Engineering, Tianjin University, Tianjin, 300072, China.

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.

No MeSH data available.


Related in: MedlinePlus