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Abscisic Acid Uridine Diphosphate Glucosyltransferases Play a Crucial Role in Abscisic Acid Homeostasis in Arabidopsis

Ting Dong, Zheng-Yi Xu, Youngmin Park, Dae Heon Kim, Yongjik Lee, Inhwan Hwang
Ting Dong
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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Zheng-Yi Xu
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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Youngmin Park
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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Dae Heon Kim
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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Yongjik Lee
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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Inhwan Hwang
Division of Integrative Biosciences and Biotechnology (T.D., Z.-Y.X., Y.P., Y.L., I.H.) and Department of Life Sciences (D.H.K., I.H.), Pohang University of Science and Technology, Pohang, 790–784 Korea
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  • ORCID record for Inhwan Hwang
  • For correspondence: ihhwang@postech.ac.kr

Published May 2014. DOI: https://doi.org/10.1104/pp.114.239210

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    Figure 1.

    Two UGT71B6 homologs, UGT71B7 and UGT71B8, reduce the cellular ABA level and are induced by exogenous ABA, NaCl, and osmotic stress. A, Phylogenetic tree of the Arabidopsis UGT71B subfamily. Members were aligned by ClustalW using the default settings for multiple alignments (gap opening of 10; gap extension cost of 0.2; 30% delay for divergent sequences; four-space gap separation distance; without end-gap separation; with residue-specific penalties; and using the Gonnet series protein-weight matrix). The aligned UGT sequences were used to construct a phylogenetic tree with Molecular Evolutionary Genetics Analysis version 5. Distance trees were created using the number of differences. The initial tree was created by maximum likelihood. To estimate the reliability of this tree, bootstrap analysis was performed with 1,000 replicates by using the same weighting parameters as those used in the initial analysis. B, Effects of three UGTs on the expression of RD29Ap:LUC in protoplasts. Protoplasts from wild-type plants were transformed with three plasmids encoding effector, reporter, and normalizer, and the transcript level of the reporter LUC was examined by qRT-PCR. UGT71B6:GFP, UGT71B7:GFP, UGT71B8:GFP, and UGT73B1:GFP were used as effectors; RD29Ap:LUC was used as a reporter; and UBQ10p:GUS was used as a normalizer. GFP alone was used as a control for the effector. GUS was used as an internal control for qRT-PCR analysis. Error bars indicate sd (n = 3). C, Induction of UGT71B6, UGT71B7, and UGT71B8 by exogenous ABA, NaCl, and mannitol. Total RNA was prepared from wild-type (WT) plants that had been treated with 100 μm ABA, 100 mm NaCl, or 300 mm mannitol for 1 h and used for qRT-PCR analysis. ACT2 was used as an internal control. Error bars indicate sd (n = 3).

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    Figure 2.

    Three GFP-tagged UGT homologs, UGT71B6:GFP, UGT71B7:GFP, and UGT71B8:GFP, are soluble proteins that localize to the cytosol. A, Localization of UGT71B6:GFP, UGT71B7:GFP, and UGT71B8:GFP. Protoplasts from UGT71B6:GFP, UGT71B7:GFP, and UGT71B8:GFP transgenic plants were isolated, and the localization of the proteins was examined. Bars = 20 μm. B, Fractionation of UGT71B6:GFP, UGT71B7:GFP, and UGT71B8:GFP. Protein extracts from plants expressing UGT71B6:GFP, UGT71B7:GFP, or UGT71B8:GFP were separated into soluble and membrane fractions by ultracentrifugation, and these fractions were analyzed by western blotting using anti-GFP antibody. As a control for the fractionation, AALP was detected using anti-AALP antibody. T, Total protein extracts from plants; S, soluble fraction; P, membrane fraction. [See online article for color version of this figure.]

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    Figure 3.

    UGT RNAi plants display hypersensitivity to exogenous ABA during germination. A, Defect in vegetative growth of RNAi plants. Plants were grown on 1/2 MS plates supplemented with 30 μm Dex. Images were taken 3 weeks after planting. B to D, Germination rates of VC, RNAi-31, and RNAi-42 plants. Seeds were planted on 1/2 MS plates supplemented with 30 μm Dex or with 30 μm Dex and 1 μm ABA. B, Images of germinating plants were taken 7 d after planting. C, Cotyledon emergence was counted at the indicated time points. D, Radicle emergence was counted 4 d after planting. For each type of plant, 50 seeds were used in a triplicate experiment. Error bars indicate sd (n = 3). E and F, Postgermination phenotypes of UGT RNAi plants. VC, RNAi-31, and RNAi-42 plants were grown on 1/2 MS plates for 5 d and transferred to 1/2 MS plates supplemented with 30 μm Dex with or without 10 μm ABA or 200 mm mannitol. E, Images were taken 2 weeks after transfer. F, Quantification of the postgermination growth rate. To quantify the growth rate, fresh weight and primary root length were measured 2 weeks after transplantation. Three independent experiments were performed with 20 plants per experiment. Error bars indicate sd (n = 60). G, Water loss of UGT RNAi plants. The aerial parts of VC, RNAi-31, and RNAi-42 plants grown on 1/2 MS plates supplemented with 30 μm Dex for 2 weeks were excised and exposed to the 30% relative humidity condition for the indicated period of time. To quantify water loss, the weight of the plant tissues was measured at the indicated time points. Error bars indicate sd (n = 30). H, Transcript levels of ABA-inducible genes in UGT RNAi transgenic plants. Total RNA was isolated from RNAi-42 transgenic plants treated with or without 30 μm Dex for 10 h, and the ABA-inducible genes RD29A, RD29B, COR47, and RAB18 were examined by qRT-PCR. The cytokinin-inducible genes ARR6 and ARR15 were included as negative controls. ACT2 was used as an internal control. Error bars indicate sd (n = 3).

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    Figure 4.

    Ectopic expression of UGT71B6 aggravates the ABA-deficient phenotype of atbg1 mutant plants. A, Germination rates of wild-type (WT), atbg1, UGT71B6:GFP/atbg1-4.3, and UGT71B6:GFP/atbg1-8.4 seeds. Seeds were planted on 1/2 MS plates, and the germination rate was determined at the indicated time points. For each plant line, 50 seeds were used in a triplicate experiment. Error bars indicate sd (n = 3). B, Effects of NaCl stress on germination rates of wild-type, atbg1, UGT71B6:GFP/atbg1-4.3, and UGT71B6:GFP/atbg1-8.4 seeds. Seeds were planted on 1/2 MS plates supplemented with 125 or 150 mm NaCl, and the germination rate was determined 4 d after planting. For each plant line, 50 seeds were used in a triplicate experiment. Error bars indicate sd (n = 3). C, Images of germinating seeds were taken 7 d after planting. D, Comparison of water loss among various types of plants. The aerial part of wild-type, UGT71B6:GFP, atbg1, and UGT71B6:GFP/atbg1-4.3 plants grown on 1/2 MS plates for 2 weeks was excised and exposed to the 30% relative humidity condition for the indicated period of time. To quantify water loss, the weight of the plant tissues was measured at the indicated time points. Error bars indicate sd (n = 20). E, Dehydration stress sensitivity among various plants. Wild-type, UGT71B6:GFP, atbg1, and UGT71B6:GFP/atbg1-4.3 plants were grown for 3 weeks on soil under normal growth conditions (watered), kept in a greenhouse without watering for 10 d, and then rewatered and grown for an additional 2 d (stressed). F, Quantification of the survival rate after dehydration stress. The survival rate was calculated from the results of five independent experiments. For each type of plant, 36 plants were used at each time. Error bars indicate sd (n = 5).

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    Figure 5.

    Ectopic expression of UGT71B6 causes a delay in the induction of osmotic stress-inducible genes. Total RNA was extracted from wild-type (WT), atbg1, UGT71B6:GFP/atbg1-4.3, and aba3 plants that had been treated for 1 h with or without 300 mm mannitol and used for qRT-PCR analysis. The transcript levels of RD29A, RAB18, and COR47 were examined using gene-specific primers. ACT2 was used as an internal control. Error bars indicate sd (n = 3).

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    Figure 6.

    Modulation of UGT71B6 expression affects the cellular ABA level. VC and RNAi-42 plants were grown on 1/2 MS plates supplemented with 30 μm Dex; wild-type (WT), atbg1, UGT71B6:GFP/atbg1-4.3, and aba3 plants were grown on 1/2 MS plates. ABA levels were measured by ELISA using anti-ABA antibody. Error bars indicate sd (n = 3).

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    Figure 7.

    The four ABA hydroxylation genes CYP707A1 to CYP707A4 are strongly induced in UGT RNAi plants. VC and RNAi-31 plants were grown for 10 d on 1/2 MS plates supplemented with 30 μm Dex and then treated for 1 h with or without dehydration stress (A) or grown in liquid medium for 10 d and treated with 30 μm Dex for 10 h followed by 1 h of treatment of 300 mm mannitol (B) or 100 mm NaCl (C). Total RNA from these plants was used for qRT-PCR analysis of CYP707A1 to CYP707A4. ACT2 was used as an internal control. Error bars indicate sd (n = 3).

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    Figure 8.

    The expression of the four ABA hydroxylation genes CYP707A1 to CYP707A4 was significantly suppressed in UGT71B6:GFP plants. Wild-type and UGT71B6:GFP plants were grown for 10 d on 1/2 MS plates and treated for 1 h with or without dehydration stress (A) or grown in liquid medium for 10 d and treated with 300 mm mannitol (B) or 100 mm NaCl (C) for 1 h. Total RNA from these plants was used for qRT-PCR of the four CYP707As. ACT2 was used as an internal control. Error bars indicate sd (n = 3).

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Abscisic Acid Uridine Diphosphate Glucosyltransferases Play a Crucial Role in Abscisic Acid Homeostasis in Arabidopsis
Ting Dong, Zheng-Yi Xu, Youngmin Park, Dae Heon Kim, Yongjik Lee, Inhwan Hwang
Plant Physiology May 2014, 165 (1) 277-289; DOI: 10.1104/pp.114.239210

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Abscisic Acid Uridine Diphosphate Glucosyltransferases Play a Crucial Role in Abscisic Acid Homeostasis in Arabidopsis
Ting Dong, Zheng-Yi Xu, Youngmin Park, Dae Heon Kim, Yongjik Lee, Inhwan Hwang
Plant Physiology May 2014, 165 (1) 277-289; DOI: 10.1104/pp.114.239210
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Plant Physiology: 165 (1)
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