A DTX/MATE-Type Transporter Facilitates Abscisic Acid Efflux and Modulates ABA Sensitivity and Drought Tolerance in Arabidopsis  Haiwen Zhang, Huifen Zhu, Yajun Pan, Yuexuan Yu, Sheng Luan, Legong Li  Molecular Plant  Volume 7, Issue 10, Pages 1522-1532 (October 2014) DOI: 10.1093/mp/ssu063 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Arabidopsis dtx50 T-DNA Insertion Mutant Phenotype. (A) Genetic map of the dtx50 insertion mutant used in this study. (B) RT–PCR results confirm dtx50 mutant and the complementary line. (C) Phenotype of young seedlings (4 weeks old). (D) The dtx50 mutant shows a growth inhibition by ABA. (E) Statistical numbers of primary root length (left), lateral root number (middle), and fresh weight (right) (mean value ± SE; n = 9) of seedlings shown in (D). (F) Seed germination assays. Left: wild-type and dtx50 seeds germinated on ½ MS medium. Right: germination rate of wild-type and dtx50 seeds on ½ MS medium supplied with different concentrations of ABA. Three independent assays were carried out with over 100 seeds each time, and an error bar was calculated. Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 DTX50 Mutation Leads to More ABA Accumulation and Up-Regulation of Several ABA Marker Genes. (A) ABA levels measured in the rosette leaves of wild-type, dtx50, and complementation line. Plants were grown on ½ MS medium and in soil for 3 weeks. (B) Q–PCR results show expression of ABF1, ABI1, Rd29B, and Rd29A in wild-type and dtx50. RNA was extracted from the rosette leaves of 3-week-old plants of wild-type and dtx50 grown on soil. Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 AtDTX50 Is an ABA Efflux Transporter. (A)AtDTX50 complements E. coli KAM3 mutant upon ABA transport. ABA was preloaded into KAM3 cells transformed with pTrc99A–DTX50 plasmid and pTrc99A vector control, respectively. The above cells were preloaded with 3H-ABA, and the 3H-ABA retained in the cells was monitored. (B)Xenopus oocytes expressed with DTX50 released more ABA. ABA was injected into the oocytes expressed DTX50 and control, and ABA release in the buffer was detected using an ABA ELISA kit. (C) The dtx50 mutant protoplast cells release less ABA than wild-type and complementation line. Protoplast cells were incubated in a buffer containing 4.5 nM ABA, then the ABA release was detected. Relative speed of ABA release was measured in dtx50, wild-type, and complementation line. The initial ABA level in each cell line was taken as 100%. (D) Substrate competition assays of ABA transport. 2 μM (+)-3H-ABA was loaded into KAM3 E. coli cells in the absence (C) or presence of additional three-fold (6 μM) unlabeled compounds including (+)-ABA, (–)-ABA, IAA, and GA in the buffer. Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression of AtDTX50. (A) The real-time Q–PCR results showing the expression levels in different tissues. Rt, root; RL, rosette leaf; CL, cauline leaf; St, stem; Fl, flower; Si, silique. (B) Q–PCR results on ABA-treated plants. Two-week-old seedlings grown on ½ MS medium were transferred to ½ MS medium supplied with 5 μM ABA, and treated for additional 1, 2, and 3 d. (C) GUS staining shows the expression pattern of DTX50. (a) The 3-week-old plants grown on ½ MS medium; (b) a close-up of a leaf; (c) a close-up of a root; (d,e) the cross-section of 2-week-old seedlings grown on ½ MS medium of leaf (d) and root (e); (f) seed germinated for 2 d; (g) stem; (h) silique; (i) flower; (j–m) 2-week-old seedlings grown on ½ MS medium (j) were sprayed with 100 μM ABA, and tissues were collected for GUS staining after 4h (k); (l) a guard cell expression of DTX50—picture was taken using the leaf shown in (j); (m) DTX50 is strongly induced by ABA—picture taken using leaf shown in (k). Scale bars: 5 mm in (a, g, i); 0.5 mm in (b, c); 10 μm in (d, e); 50 μm in (f); 2 mm in (h, j, k); 50 μm in (l, m). Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 DTX50::GFP Is Localized at the Plasma Membrane. (A)Arabidopsis mesophyll protoplasts were transiently transformed with DTX50pr–DTX50::GFP (native promoter driven) and 35S-GFP as a control. Bar = 5 μM. (B) Onion epidermal cells were transiently transformed with 35S–DTX50::GFP (35S promoter driven) and 35S-GFP as a control. Bar = 50 μm Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 The dtx50 Mutant Showed Better Tolerance under Drought Stress. (A) The dtx50 mutant is more tolerant to drought stress. Four-week-old plants were grown in the same pot under short-day conditions in a greenhouse (upper left), water was withheld for 20 d (upper right) and 28 d (lower left), and plants were then rehydrated (lower right). (B) Time course of water loss from the detached leaves of 4-week-old wild-type and dtx50. The water loss is shown as a percentage of the initial fresh weight at indicated intervals. Three independent experiments were carried out, and at least three plants were calculated each time. The mean values of three measurements are shown with error bars (SE). (C) Stomatal aperture of wild-type and dtx50 under the presence of 1 μM ABA. Leaves were immersed in 10 mM KCl-MES open buffer for over 3h under 130 μmol m−2 s−1 white light, then ABA was added at a final concentration of 1 μM. Epidermal strips were peeled and pictures were taken under a microscope to calculate the stomatal aperture. Three independent experiments were carried out, and about 60 guard cells were calculated each time. Molecular Plant 2014 7, 1522-1532DOI: (10.1093/mp/ssu063) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions