Limits...
Arabidopsis seed-specific vacuolar aquaporins are involved in maintaining seed longevity under the control of ABSCISIC ACID INSENSITIVE 3.

Mao Z, Sun W - J. Exp. Bot. (2015)

Bottom Line: TIP3;1 and TIP3;2 promoters could be activated by ABI3 in the presence of abscisic acid (ABA) in Arabidopsis protoplasts.TIP3 proteins were detected in the protoplasts transiently expressing ABI3 and in ABI3-overexpressing seedlings when treated with ABA.Furthermore, ABI3 directly binds to the RY motif of the TIP3 promoters.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Fenglin Road 300, Shanghai, 200032, People's Republic of China.

No MeSH data available.


Related in: MedlinePlus

ABI3 binds to the TIP3 promoters through their RY motifs. (A) Diagram of the TIP3;1 and TIP3;2 promoter regions. The RY motifs are shown in black boxes. (B) Transient expression assay with mutant TIP3;1 and TIP3;2 promoters. The TIP3;1 mutant promoter contains mutations in the RY1, RY2, and RY3 motifs. The TIP3;2 mutant promoter contains a mutation in the RY motif. Protoplasts were transformed with the effector plasmid containing ABI3 and treated with 5 μM ABA. Values are means ±SD, n=3. (C) Relative expression levels of the GUS reporter gene driven by the TIP3;1 promoters with or without mutations in the RY motifs. RNA was extracted from seeds of 10 independent transgenic lines carrying the WT or mutant promoters fused to GUS. Each point represents the mean of three replicates of one transgenic line, and SD values were omitted for clarity. (D and F) EMSA demonstrating the binding of the B3 domain of ABI3 to the RY2 element in the TIP3;1 promoter (D) or the RY element in the TIP3;2 promoter (F). The numbers indicate the amount of B3 domain of ABI3 protein used in the assays. (E and G) Binding specificity of ABI3 protein to the RY2 element in the TIP3;1 promoter (E) and the RY element in the TIP3;2 promoter (G). Binding specificity was demonstrated with competition experiments by adding 40- or 200-fold excessive non-labelled WT or mutant probes. Arrows indicate the gel retardation complexes formed between RY elements and the B3 domain of ABI3 protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4507774&req=5

Figure 6: ABI3 binds to the TIP3 promoters through their RY motifs. (A) Diagram of the TIP3;1 and TIP3;2 promoter regions. The RY motifs are shown in black boxes. (B) Transient expression assay with mutant TIP3;1 and TIP3;2 promoters. The TIP3;1 mutant promoter contains mutations in the RY1, RY2, and RY3 motifs. The TIP3;2 mutant promoter contains a mutation in the RY motif. Protoplasts were transformed with the effector plasmid containing ABI3 and treated with 5 μM ABA. Values are means ±SD, n=3. (C) Relative expression levels of the GUS reporter gene driven by the TIP3;1 promoters with or without mutations in the RY motifs. RNA was extracted from seeds of 10 independent transgenic lines carrying the WT or mutant promoters fused to GUS. Each point represents the mean of three replicates of one transgenic line, and SD values were omitted for clarity. (D and F) EMSA demonstrating the binding of the B3 domain of ABI3 to the RY2 element in the TIP3;1 promoter (D) or the RY element in the TIP3;2 promoter (F). The numbers indicate the amount of B3 domain of ABI3 protein used in the assays. (E and G) Binding specificity of ABI3 protein to the RY2 element in the TIP3;1 promoter (E) and the RY element in the TIP3;2 promoter (G). Binding specificity was demonstrated with competition experiments by adding 40- or 200-fold excessive non-labelled WT or mutant probes. Arrows indicate the gel retardation complexes formed between RY elements and the B3 domain of ABI3 protein.

Mentions: The B3 domain of ABI3 has DNA binding specificity and recognizes the RY motif (CATGCA) (Suzuki et al., 1997). Promoter sequence analysis showed that TIP3;1 and TIP3;2 promoters possess three and one potential RY motif, respectively (Fig. 6A). To determine whether RY motifs are critical for the activation of TIP3 promoters by ABI3, transient expression assays were performed with the TIP3;1 promoter mutated in RY1, RY2, and RY3 motifs and the TIP3;2 promoter mutated in the RY motif, respectively. Mutations in RY motifs caused reduction of both TIP3 promoter activities (Fig. 6B), suggesting that the RY motifs are required for the promoter activities of TIP3 genes. To determine further which RY motif is important for TIP3;1 promoter activity, promoters containing mutations in the RY motif were fused to the GUS reporter gene and transgenic Arabidopsis plants were generated. GUS expression in transgenic seeds of different lines was determined by qRT-PCR. Mutation of the RY3 or RY1 motif caused a slight reduction in GUS expression, while mutation of the RY2 motif caused a more significant reduction in GUS expression (Fig. 6C). Additional mutations in RY3 or in the RY1 and RY3 motifs did not further reduce the activities of promoters containing a mutation in the RY2 motif (Fig. 6C). This result suggests that the RY2 motif is essential for TIP3;1 expression in seeds.


Arabidopsis seed-specific vacuolar aquaporins are involved in maintaining seed longevity under the control of ABSCISIC ACID INSENSITIVE 3.

Mao Z, Sun W - J. Exp. Bot. (2015)

ABI3 binds to the TIP3 promoters through their RY motifs. (A) Diagram of the TIP3;1 and TIP3;2 promoter regions. The RY motifs are shown in black boxes. (B) Transient expression assay with mutant TIP3;1 and TIP3;2 promoters. The TIP3;1 mutant promoter contains mutations in the RY1, RY2, and RY3 motifs. The TIP3;2 mutant promoter contains a mutation in the RY motif. Protoplasts were transformed with the effector plasmid containing ABI3 and treated with 5 μM ABA. Values are means ±SD, n=3. (C) Relative expression levels of the GUS reporter gene driven by the TIP3;1 promoters with or without mutations in the RY motifs. RNA was extracted from seeds of 10 independent transgenic lines carrying the WT or mutant promoters fused to GUS. Each point represents the mean of three replicates of one transgenic line, and SD values were omitted for clarity. (D and F) EMSA demonstrating the binding of the B3 domain of ABI3 to the RY2 element in the TIP3;1 promoter (D) or the RY element in the TIP3;2 promoter (F). The numbers indicate the amount of B3 domain of ABI3 protein used in the assays. (E and G) Binding specificity of ABI3 protein to the RY2 element in the TIP3;1 promoter (E) and the RY element in the TIP3;2 promoter (G). Binding specificity was demonstrated with competition experiments by adding 40- or 200-fold excessive non-labelled WT or mutant probes. Arrows indicate the gel retardation complexes formed between RY elements and the B3 domain of ABI3 protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4507774&req=5

Figure 6: ABI3 binds to the TIP3 promoters through their RY motifs. (A) Diagram of the TIP3;1 and TIP3;2 promoter regions. The RY motifs are shown in black boxes. (B) Transient expression assay with mutant TIP3;1 and TIP3;2 promoters. The TIP3;1 mutant promoter contains mutations in the RY1, RY2, and RY3 motifs. The TIP3;2 mutant promoter contains a mutation in the RY motif. Protoplasts were transformed with the effector plasmid containing ABI3 and treated with 5 μM ABA. Values are means ±SD, n=3. (C) Relative expression levels of the GUS reporter gene driven by the TIP3;1 promoters with or without mutations in the RY motifs. RNA was extracted from seeds of 10 independent transgenic lines carrying the WT or mutant promoters fused to GUS. Each point represents the mean of three replicates of one transgenic line, and SD values were omitted for clarity. (D and F) EMSA demonstrating the binding of the B3 domain of ABI3 to the RY2 element in the TIP3;1 promoter (D) or the RY element in the TIP3;2 promoter (F). The numbers indicate the amount of B3 domain of ABI3 protein used in the assays. (E and G) Binding specificity of ABI3 protein to the RY2 element in the TIP3;1 promoter (E) and the RY element in the TIP3;2 promoter (G). Binding specificity was demonstrated with competition experiments by adding 40- or 200-fold excessive non-labelled WT or mutant probes. Arrows indicate the gel retardation complexes formed between RY elements and the B3 domain of ABI3 protein.
Mentions: The B3 domain of ABI3 has DNA binding specificity and recognizes the RY motif (CATGCA) (Suzuki et al., 1997). Promoter sequence analysis showed that TIP3;1 and TIP3;2 promoters possess three and one potential RY motif, respectively (Fig. 6A). To determine whether RY motifs are critical for the activation of TIP3 promoters by ABI3, transient expression assays were performed with the TIP3;1 promoter mutated in RY1, RY2, and RY3 motifs and the TIP3;2 promoter mutated in the RY motif, respectively. Mutations in RY motifs caused reduction of both TIP3 promoter activities (Fig. 6B), suggesting that the RY motifs are required for the promoter activities of TIP3 genes. To determine further which RY motif is important for TIP3;1 promoter activity, promoters containing mutations in the RY motif were fused to the GUS reporter gene and transgenic Arabidopsis plants were generated. GUS expression in transgenic seeds of different lines was determined by qRT-PCR. Mutation of the RY3 or RY1 motif caused a slight reduction in GUS expression, while mutation of the RY2 motif caused a more significant reduction in GUS expression (Fig. 6C). Additional mutations in RY3 or in the RY1 and RY3 motifs did not further reduce the activities of promoters containing a mutation in the RY2 motif (Fig. 6C). This result suggests that the RY2 motif is essential for TIP3;1 expression in seeds.

Bottom Line: TIP3;1 and TIP3;2 promoters could be activated by ABI3 in the presence of abscisic acid (ABA) in Arabidopsis protoplasts.TIP3 proteins were detected in the protoplasts transiently expressing ABI3 and in ABI3-overexpressing seedlings when treated with ABA.Furthermore, ABI3 directly binds to the RY motif of the TIP3 promoters.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Fenglin Road 300, Shanghai, 200032, People's Republic of China.

No MeSH data available.


Related in: MedlinePlus