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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 regulates the expression of TIP3 genes. (A and B) qRT-PCR and immunoblot analysis of the expression of TIP3 genes in abi3-6 and fus3-3 seeds. Values in (A) are means ±SD, n=3. (C) The TIP3;1 and TIP3;2 promoters are activated by ABI3 when treated with ABA in a transient expression assay. Values are means ±SD, n=3. Protoplasts transformed with empty pGREENII 62-SK vector were used as a control; 5 μM ABA was supplied in the ABA treatment. (D) qRT-PCR analysis of TIP3 and EM1 transcript levels in the WT (Col) and a transgenic line ectopically expressing ABI3 (Pro35S:ABI3). For ABA treatment, 3-week-old seedlings grown on MS medium were transferred to MS medium supplemented with 50 μM ABA for 3 d. PP2A was used as an endogenous control. (E) Immunoblot analysis of TIP3s and TIP1s in protoplasts transiently expressing ABI3 or FUS3 in the presence of 5 μM ABA. Detection of actin by an antibody was used as a loading control. (F) Immunoblot analysis of TIP3s in seedlings of WT and Pro35S:ABI3 transgenic Arabidopsis. ABA treatment was performed as described in (D). DS, dry mature seeds.
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Figure 5: ABI3 regulates the expression of TIP3 genes. (A and B) qRT-PCR and immunoblot analysis of the expression of TIP3 genes in abi3-6 and fus3-3 seeds. Values in (A) are means ±SD, n=3. (C) The TIP3;1 and TIP3;2 promoters are activated by ABI3 when treated with ABA in a transient expression assay. Values are means ±SD, n=3. Protoplasts transformed with empty pGREENII 62-SK vector were used as a control; 5 μM ABA was supplied in the ABA treatment. (D) qRT-PCR analysis of TIP3 and EM1 transcript levels in the WT (Col) and a transgenic line ectopically expressing ABI3 (Pro35S:ABI3). For ABA treatment, 3-week-old seedlings grown on MS medium were transferred to MS medium supplemented with 50 μM ABA for 3 d. PP2A was used as an endogenous control. (E) Immunoblot analysis of TIP3s and TIP1s in protoplasts transiently expressing ABI3 or FUS3 in the presence of 5 μM ABA. Detection of actin by an antibody was used as a loading control. (F) Immunoblot analysis of TIP3s in seedlings of WT and Pro35S:ABI3 transgenic Arabidopsis. ABA treatment was performed as described in (D). DS, dry mature seeds.

Mentions: The transcription factor ABI3 is involved in seed desiccation tolerance and seed longevity (Ooms et al., 1993; Tesnier et al., 2002). TIP3 genes are seed-specific genes during seed maturation, and tip3 knockdown mutant seeds exhibit a decrease of seed longevity similar to the seed longevity phenotype of abi3-1 and abi3-7. It was hypothesized that TIP3s may maintain seed longevity under the expressional regulation of ABI3. To test whether the seed-specific transcription factors ABI3 or FUS3 are involved in the regulation of TIP3 gene expression in seeds, the presence of TIP3 gene transcripts in the corresponding mutant seeds was investigated. As expected, TIP3;1 and TIP3;2 transcripts were not detectable in abi3-6 mutant seeds (Fig. 5A). The abi3-6 allele contains a deletion in ABI3 which causes a premature stop codon and leads to translation of a short form protein containing only the A1 domain but not the B1, B2, and B3 domains. The expression levels of TIP3 genes in the fus3-3 mutant decreased ~50% compared with those of the WT (Col) (Fig. 5A). The protein levels of TIP3s decreased significantly in the abi3-6 and fus3-3 mutants (Fig. 5B). The expression levels of ABI3 in abi3-6 and fus3-3 mutant seeds were also analysed. In fus3-3 seeds, ABI3 expression decreased to 50% compared with the WT (Supplementary Fig. S5 at JXB online). Therefore, the reduction of TIP3 gene expression in fus3-3 correlates with decreased expression of ABI3 in fus3-3 mutant seeds. the expression of TIP3 genes was also analysed in other alleles of ABI3, namely abi3-1, abi3-4, and abi3-8. The amount of TIP3 transcripts and the abundance of proteins were reduced to varying degrees in these abi3 mutants (Supplementary Fig. S6). TIP3;1 promoter activity was also reduced in the abi3-6 mutant. No green fluorescent protein (GFP) fluorescence and GFP protein expression could be detected in isolated abi3-6 embryos transformed with ProTIP3;1:GFP (Supplementary Fig. S7). Taken together, these results suggest that ABI3 is required for TIP3 gene expression and protein accumulation in mature 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 regulates the expression of TIP3 genes. (A and B) qRT-PCR and immunoblot analysis of the expression of TIP3 genes in abi3-6 and fus3-3 seeds. Values in (A) are means ±SD, n=3. (C) The TIP3;1 and TIP3;2 promoters are activated by ABI3 when treated with ABA in a transient expression assay. Values are means ±SD, n=3. Protoplasts transformed with empty pGREENII 62-SK vector were used as a control; 5 μM ABA was supplied in the ABA treatment. (D) qRT-PCR analysis of TIP3 and EM1 transcript levels in the WT (Col) and a transgenic line ectopically expressing ABI3 (Pro35S:ABI3). For ABA treatment, 3-week-old seedlings grown on MS medium were transferred to MS medium supplemented with 50 μM ABA for 3 d. PP2A was used as an endogenous control. (E) Immunoblot analysis of TIP3s and TIP1s in protoplasts transiently expressing ABI3 or FUS3 in the presence of 5 μM ABA. Detection of actin by an antibody was used as a loading control. (F) Immunoblot analysis of TIP3s in seedlings of WT and Pro35S:ABI3 transgenic Arabidopsis. ABA treatment was performed as described in (D). DS, dry mature seeds.
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Figure 5: ABI3 regulates the expression of TIP3 genes. (A and B) qRT-PCR and immunoblot analysis of the expression of TIP3 genes in abi3-6 and fus3-3 seeds. Values in (A) are means ±SD, n=3. (C) The TIP3;1 and TIP3;2 promoters are activated by ABI3 when treated with ABA in a transient expression assay. Values are means ±SD, n=3. Protoplasts transformed with empty pGREENII 62-SK vector were used as a control; 5 μM ABA was supplied in the ABA treatment. (D) qRT-PCR analysis of TIP3 and EM1 transcript levels in the WT (Col) and a transgenic line ectopically expressing ABI3 (Pro35S:ABI3). For ABA treatment, 3-week-old seedlings grown on MS medium were transferred to MS medium supplemented with 50 μM ABA for 3 d. PP2A was used as an endogenous control. (E) Immunoblot analysis of TIP3s and TIP1s in protoplasts transiently expressing ABI3 or FUS3 in the presence of 5 μM ABA. Detection of actin by an antibody was used as a loading control. (F) Immunoblot analysis of TIP3s in seedlings of WT and Pro35S:ABI3 transgenic Arabidopsis. ABA treatment was performed as described in (D). DS, dry mature seeds.
Mentions: The transcription factor ABI3 is involved in seed desiccation tolerance and seed longevity (Ooms et al., 1993; Tesnier et al., 2002). TIP3 genes are seed-specific genes during seed maturation, and tip3 knockdown mutant seeds exhibit a decrease of seed longevity similar to the seed longevity phenotype of abi3-1 and abi3-7. It was hypothesized that TIP3s may maintain seed longevity under the expressional regulation of ABI3. To test whether the seed-specific transcription factors ABI3 or FUS3 are involved in the regulation of TIP3 gene expression in seeds, the presence of TIP3 gene transcripts in the corresponding mutant seeds was investigated. As expected, TIP3;1 and TIP3;2 transcripts were not detectable in abi3-6 mutant seeds (Fig. 5A). The abi3-6 allele contains a deletion in ABI3 which causes a premature stop codon and leads to translation of a short form protein containing only the A1 domain but not the B1, B2, and B3 domains. The expression levels of TIP3 genes in the fus3-3 mutant decreased ~50% compared with those of the WT (Col) (Fig. 5A). The protein levels of TIP3s decreased significantly in the abi3-6 and fus3-3 mutants (Fig. 5B). The expression levels of ABI3 in abi3-6 and fus3-3 mutant seeds were also analysed. In fus3-3 seeds, ABI3 expression decreased to 50% compared with the WT (Supplementary Fig. S5 at JXB online). Therefore, the reduction of TIP3 gene expression in fus3-3 correlates with decreased expression of ABI3 in fus3-3 mutant seeds. the expression of TIP3 genes was also analysed in other alleles of ABI3, namely abi3-1, abi3-4, and abi3-8. The amount of TIP3 transcripts and the abundance of proteins were reduced to varying degrees in these abi3 mutants (Supplementary Fig. S6). TIP3;1 promoter activity was also reduced in the abi3-6 mutant. No green fluorescent protein (GFP) fluorescence and GFP protein expression could be detected in isolated abi3-6 embryos transformed with ProTIP3;1:GFP (Supplementary Fig. S7). Taken together, these results suggest that ABI3 is required for TIP3 gene expression and protein accumulation in mature 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