AtSWEET13 and AtSWEET14 regulate gibberellin-mediated physiological processes

Abstract

Transmembrane transport of plant hormones is required for plant growth and development. Despite reports of a number of proteins that can transport the plant hormone gibberellin (GA), the mechanistic basis for GA transport and the identities of the transporters involved remain incomplete. Here, we provide evidence that Arabidopsis SWEET proteins, AtSWEET13 and AtSWEET14, which are members of a family that had previously been linked to sugar transport, are able to mediate cellular GA uptake when expressed in yeast and oocytes. A double sweet13 sweet14 mutant has a defect in anther dehiscence and this phenotype can be reversed by exogenous GA treatment. In addition, sweet13 sweet14 exhibits altered long distant transport of exogenously applied GA and altered responses to GA during germination and seedling stages. These results suggest that AtSWEET13 and AtSWEET14 may be involved in modulating GA response in Arabidopsis.

Figure 1. Identification of Arabidopsis SWEET proteins as candidate GA transporters.

(a) A phylogenetic tree of 17 Arabidopsis SWEET proteins was generated by ClustalX using the neighbour-joining method and displayed using Njplot. The scale bar indicates a branch length corresponding to 0.5 substitutions per site. (b) The effects of six closely related AtSWEET proteins on the interaction between GID1a and GAI determined by Y2H assays using HIS3 as a selection marker. AtSWEET9, AtSWEET10, AtSWEET11, AtSWEET12, AtSWEET13 or AtSWEET14 was expressed in yeast containing the Y2H system with BD-GID1a and AD-GAI, and grown on selection media (SD; -Leu, -Trp, -Ura, -His) containing GA₁ (0.1 μM), GA₃ (0.1 μM) or GA₄ (1 nM) for 7 days. Yeast transformed with an empty vector was used as a control. 1 × 10⁴ cells were inoculated on the media. (c) The effects of six closely related AtSWEET proteins on the interaction between GID1a and GAI were determined by Y2H assays based on the expression of the lacZ marker. Units of β-galactosidase (β-gal) activity represent 1,000 × OD₄₂₀/min/OD₆₀₀ cells. Values are means±s.d. of three biological replicates.

Figure 2. AtSWEET13 and AtSWEET14 transport GA.

(a) GA transport activities of AtSWEET13 and AtSWEET14 in yeast. Yeast cells expressing AtSWEET13 or AtSWEET14 were incubated with 10 μM GA₃ in the presence 100 mM glucose at pH 5.8. Yeast transformed with an empty vector was used as a control. (b) Plant hormone transport activity of AtSWET13 and AtSWEET14. Yeast cells expressing AtSWEET13 or AtSWEET14 were incubated with 10 μM IAA, ABA, JA or JA-Ile in the presence 100 mM glucose at pH 5.8. Yeast transformed with an empty vector was used as a control. (c) GA transport activities of AtSWEET13 and AtSWEET14 in Xenopus oocytes. Oocytes injected with AtSWEET13 or AtSWEET14 cRNA were incubated for 24 h in Kulori medium-based buffer (pH 5.0) containing 100 μM GA₃. As a control, water was injected into the oocytes. AtNPF2.10/GTR1 cRNA was also injected for comparison. Values are means±s.d. of four biological replicates with two oocytes. (d) Substrate preferences of AtSWEET13 and AtSWEET14 for different GA species. Yeast cells expressing AtSWEET13 or AtSWEET14 were incubated with a mixture of 11 GAs (5 μM each) in the presence of 100 mM glucose at pH 5.8. Yeast transformed with an empty vector was used as a control. For (a,b,d), compounds taken into cells after incubation for 5 min were analysed by LC–MS/MS. Values are means±s.d. of three biological replicates.

Figure 3. Spatial expression patterns of AtSWEET13 and AtSWEET14.

(a–f) GUS activities of pAtSWEET13:GUS transgenic plants. (g–l) GUS activities of pAtSWEET14:GUS transgenic plants. (a,g), flower buds; (b,h), anthers; (c,i), shoots of 2-week-old seedlings; (d,j), roots of 2-week-old seedlings; (e,k), junctions of stems and petioles of 2-week-old seedlings; (f,l) embryos at 15 days after flowering. Arrowheads in e and k show dot-like staining at the junctions of stems and petioles. Scale bars: 1 mm for a,c,g and i, 0.2 mm for b,f,h and l, 0.5 mm for d,e,j and k.

Figure 4. Subcellular localization of AtSWEET13 and AtSWEET14.

(a) GFP fluorescence in p35S:AtSWEET13-GFP transgenic plants. (b) GFP fluorescence in p35S:AtSWEET14-GFP transgenic plants. For (a,b), roots of 1-week-old seedlings treated with water (H₂O) or 20% (w/v) sucrose were used for observations. Upper panels are bright-field images. Middle panels are images of GFP fluorescence. Lower panels are merged images of bright-field and GFP fluorescence. Scale bars, 20 μm.

Figure 5. Phenotypes of sweet13 sweet14 in flowers and siliques.

(a) Representative siliques from wild type (WT), sweet13 (13), sweet14 (14) and sweet13 sweet14 (13 14). Positions of the siliques from the bottom are indicated on the left side of the photo. Scale bar, 1 cm. (b) Numbers of seeds per siliques in wild type (WT), sweet13 (13), sweet14 (14) and sweet13 sweet14 (13 14). Averages of eight siliques are shown with standard deviations. The seed number of siliques at different positions varied significantly; *P<0.05, **P<0.001; Tukey–Kramer test. (c) Representative photos of flowers and anthers of wild type (WT) and sweet13 sweet14. Scale bar, 1 mm. (d) Restoration of the sweet13 sweet14 phenotype by GA₃ treatment. Excised flower buds of sweet13 sweet14 were incubated with water (13 14) or 100 μM GA₃ (13 14+GA₃) and anthers were observed after 1 day. Wild type flower buds incubated with water were also observed for comparison (WT). Scale bar, 0.1 mm.

Figure 6. Phenotypes of sweet13 sweet14 in seeds and seedlings.

(a) Dry seeds of wild type (WT), sweet13 (13), sweet14 (14), and sweet13 sweet14 (13 14). Scale bar, 0.5 mm. (b) Phenotypes of wild type (WT), sweet13 (13), sweet14 (14), sweet13 sweet14 (13 14), ga3ox1 (3ox1), ga3ox1 sweet13 sweet14 (3ox1 13 14), ga20ox1 (20ox1), and ga20ox1 sweet13 sweet14 (20ox1 13 14). Upper panel: weight for 100 dry seeds was measured independently three times for each genotype and the average values are shown with standard deviations. Middle panel: root length of 8-day-old seedlings grown on Murashige and Skoog media without sugars (averages of 40–50 seedlings) is shown with ±s.d.. Lower panel: shoot dry weight of 13–20 seedlings that were grown for 8 days on Murashige and Skoog media without sugars was measured and the weight per shoot was calculated. Values are means±s.d. of three biological replicates. **P<0.001; ns, not significant (P>0.05); Tukey–Kramer test. (c) Seedlings of (WT), sweet13 (13), sweet14 (14), and sweet13 sweet14 (13 14) grown for 8 days on Murashige and Skoog media without sugars. Scale bar, 1 cm. (d) Germination of wild type (WT) and sweet13 sweet14 (13 14) in the presence or absence of 0.5 μM ABA or 10 μM paclobutrazol (Pac). Germination rates of approximately 50 seeds were scored every day and averages of three biological replicates are shown with standard deviations. Cotyledon greening was the indicator of germination.

Figure 7. Transport of exogenously applied GA from roots to shoots.

The primary root tips of 8-day-old wild-type (WT) and sweet13 sweet14 (13 14) seedlings were spotted with 2μl of 10 μM GA₃. After incubation for 0, 1, 3 and 6 h, the fragment ion with a m/z of 221.2 derived from the molecular ion with a m/z of 345.2 was detected from shoots by LC–MS/MS. Peak areas relative to those of wild type before the treatment (0 h) are shown as means of three biological replicates with standard deviations. *P<0.05; NS, not significant (P>0.05); Student's t-test.