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

Abstract

The tonoplast intrinsic proteins TIP3;1 and TIP3;2 are specifically expressed during seed maturation and localized to the seed protein storage vacuole membrane. However, the function and physiological roles of TIP3s are still largely unknown. The seed performance of TIP3 knockdown mutants was analysed using the controlled deterioration test. The tip3;1/tip3;2 double mutant was affected in seed longevity and accumulated high levels of hydrogen peroxide compared with the wild type, suggesting that TIP3s function in seed longevity. The transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3) is known to be involved in seed desiccation tolerance and seed longevity. TIP3 transcript and protein levels were significantly reduced in abi3-6 mutant seeds. 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. Therefore, seed-specific TIP3s may help maintain seed longevity under the expressional control of ABI3 during seed maturation and are members of the ABI3-mediated seed longevity pathway together with small heat shock proteins and late embryo abundant proteins.

Keywords: ABI3; Arabidopsis; TIP3.; hydrogen peroxide; seed longevity.

Fig. 1.

TIP3 genes are specifically expressed during seed maturation. (A and B) Expression analysis of TIP3;1 and TIP3;2 during seed development (A) and seed germination (B) in Arabidopsis. qRT-PCR analysis of TIP3;1 and TIP3;2 transcript abundance during seed development and seed germination. The relative expression level of each gene was normalized with four reference genes, and calculated according to the geNorm 3.5 manual. Values are means ±SD, n=3. DPA, days post-anthesis. (C and D) Immunoblot analysis of TIP3s during seed development (C) and seed germination (D). The same amounts of proteins separated by SDS–PAGE were stained with Coomassie Brilliant Blue and used as a loading control.

Fig. 2.

Identification of tip3;1 and tip3;2 T-DNA insertion mutants and three TIP3;1-RNAi transgenic lines (TIP3;1-RNAi/tip3;2) in the tip3;2 mutant background. (A) Schematic representation of the tip3;1 and tip3;2 T-DNA insertion mutant lines. A triangle indicates the position of the T-DNA insertion, and the arrow indicates its orientation. The genomic sequences corresponding to the coding region (black boxes), untranslated region (grey boxes), and introns (black lines) are indicated. The positions of the primers (31LP, 31RP, 32LP, and 32RP) used for PCR analysis of the tip3;1 and tip3;2 T-DNA insertion mutants, respectively, are also indicated. (B) PCR analysis of genomic DNA of Col, tip3;1, tip3;2, and tip3;1/tip3;2. LP, left primer; RP, right primer; LB, T-DNA left border primer. (C) Schematic representation of the construct used for the suppression of TIP3;1 in Arabidopsis seeds. RNAi technology was used with a segment of the TIP3;1 gene driven by the seed-specific TIP3;1 promoter. (D) qRT-PCR analysis of TIP3;1, TIP3;2, and ACT7 transcript abundance in mature seeds of Col, mutants, and RNAi lines. PP2A was used as the endogenous control, and the transcript abundance of TIP3;1, TIP3;2, and ACT7 was quantified by comparisons with that of PP2A. Values are means ±SD, n=3. (E) Immunoblot analysis of TIP3s in mature seeds from Col, tip3 mutants, and three TIP3;1-RNAi/tip3;2 transgenic lines (R3, R7, and R8). HSP17.6, which is expressed in mature seeds, was used as a loading control. (This figure is available in colour at JXB online.)

Fig. 3.

Natural and artificial seed ageing assays showing that TIP3 genes are involved in maintaining seed longevity. (A) Germination and growth of seeds from Col, tip3;2, and three lines of TIP3;1-RNAi/tip3;2 stored for 2 weeks or 18 months. The photographs were taken 6 d after seed imbibition. (B) Germination percentages of seeds 6 d after imbibition. (C) Germination percentages of tip3 mutants and TIP3;1-RNAi/tip3;2 transgenic seeds submitted to a CDT for 1–7 d. The germination percentages were counted 7 d after imbibition. (D) Germination percentages of Ler and abi3-1 seeds submitted to a CDT for 1–7 d. The germination percentages were counted 7 d after imbibition. (E) Germination percentages of tip3 mutants and TIP3;1-RNAi/tip3;2 transgenic seeds after a 4 d CDT. Germination percentages were counted at different time points after imbibition. (F) Germination percentages of Ler and abi3-1 seeds after a 4 d CDT. Germination percentages were counted at different time points after imbibition. Values are the means ±SD of four technical replicates with 100 seeds per replicate. In (C–E) seeds used for germination tests were harvested at the same time and stored for 2 weeks prior to the experiment.

Fig. 4.

TIP3s are involved in maintaining seed viability during the CDT. (A) Seed viability after a 0–7 d CDT. Seed viability was analysed by tetrazolium staining. (B) Staining of H₂O₂ in the embryos of Col and tip3;1/tip3;2 seeds submitted to a CDT for 0–7 d. Seeds used for the CDT were harvested at the same time and stored for 2 weeks prior to the assay.

Fig. 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 (Pro _35S :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 Pro _35S :ABI3 transgenic Arabidopsis. ABA treatment was performed as described in (D). DS, dry mature seeds.

Fig. 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.

Fig. 7.

TIP3;2 facilitates H₂O₂ diffusion. (A) Survival test of three different yeast strains transformed with TIP3 genes on medium containing H₂O₂. Yeast strains Δdur3, Δyap1, and Δskn7 were transformed with pYX212 (or derivatives of pYX212 carrying AQP cDNAs). Yeast cells were diluted to an OD₆₀₀ of 0.1 with SD-Ura liquid medium, and 10 μl were spotted onto SD-Ura medium containing various concentrations of H₂O₂. Numbers indicate the concentration of H₂O₂ (mM). Photographs were taken 3 d after incubation at 30 °C. (B) TIP3;2 mediates H₂O₂ diffusion across the membrane in yeast. The fluorescence of CM-H₂DCFDA-loaded yeast cells transformed with pYX212 or pYX212 carrying the indicated AQP cDNAs wase measured 30min after incubation with 0, 2, or 10mM H₂O₂. Histograms represent the average increase in fluorescence after 30min incubation. Data are means ±SD, n=3.