Volume 5, Issue 5, Pages 1029-1041 (September 2012) Arabidopsis Sucrose Transporter SUT4 Interacts with Cytochrome b5-2 to Regulate Seed Germination in Response to Sucrose and Glucose  Yan Li, Ling-Li Li, Ren-Chun Fan, Chang-Cao Peng, Hai-Li Sun, Sai-Yong Zhu, Xiao-Fang Wang, Ling-Yun Zhang, Da-Peng Zhang  Molecular Plant  Volume 5, Issue 5, Pages 1029-1041 (September 2012) DOI: 10.1093/mp/sss001 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Sucrose Transporter SUT4, but Not SUT1 and SUT2, Interacts with All the Cytochrome b5 Members except for Cyb5-5 in the Yeast Two-Hybrid Split-Ubiquitin System. Top panels show the transformant yeast growth in the medium supplemented with histidine (+ Histidine), and the bottom panels show the transformant yeast growth in the histidine-deficient medium (– Histidine). The yeast cells co-transformed with the empty pTMBV4 and pDL2 vectors were used as a negative control (Control). The assays were repeated three times with the same results. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Interaction between SUT4 and Cyb5-2 In Vivo. (A) Immunoprecipitation assay. The total membrane proteins prepared from the GFP-tagged SUT4-transgenic plants were immunoprecipitated with anti-Cyb5-2 serum (IP: anti-Cyb5-2) and immuno-blotted with the anti-GFP serum (Blot: anti-GFP). The sample precipitated with pre-immune serum was used as a negative control (IP: preimmune serum), and the same total membrane proteins without being subjected to immunoprecipitation procedures were used as positive controls. The protein band indicated by 84 kD (56 kD of SUT4 plus 28 kD of GFP) shows the GFP-tagged SUT4. The assays were conducted by using three independent SUT4–GFP-transgenic lines and, for each transgenic line, the assay were repeated three times, which gave the same results. (B) Bimolecular fluorescence complementation (BiFC) assay. Protoplasts were transformed with the construct pair YFPN–Cyb5-2 plus SUT4–YFPC (YFPN–Cyb5/SUT4–YFPC). The protoplasts transformed with the construct pairs YFPN–Cyb5-2 plus YFPC (YFPN–Cyb5/YFPC) or YFPN plus SUT4–YFPC (YFPN/SUT4–YFPC) or YFPN plus YFPC (YFPN/YFPC) were used as negative controls. YFP, fluorescence indicating protein interaction; Auto-, chlorophyll auto-fluorescence; Bright, bright field; Merged, merged image of YFP, Auto- and Bright. The assays were repeated five times, with the same results. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Sucrose, But Not Glucose, Inhibits the Interaction between SUT4 and Cyb5-2. (A) SUT4 and Cyb5-2 interaction in the yeast two-hybrid split-ubiquitin system measured with the drop test assay. The SUT4 and Cyb5-2 co-transformed yeast cells of 8 μl were spotted at four concentrations (OD600 = 0.001, 0.005, 0.01, and 0.1) in the histidine-deficient (SD-3: -Leu, -Trp, -His) medium supplemented with 0.1% (3 mM), 0.2% (6 mM), 0.4% (12 mM), or 2% (60 mM) sucrose or with 2% (111 mM) glucose as sole carbon source. The assays were repeated three times, with the same results. (B) Growth of the yeast cells transformed only with SUT4 (pTMBV4–AtSUT4) in the SD medium (supplied with Leu, Trp, and His) supplemented with 3, 6, 12, or 60 mM sucrose as carbon source. The SUT4-transformed yeast cells of 8 μl were spotted at the same concentration (OD600 = 0.005). The assays were repeated three times, with the same results. (C) β-gal activity measured in the yeast two-hybrid split-ubiquitin system. The SUT4 and Cyb5-2 co-transformed yeast cells were grown in SD medium (supplied with Leu, Trp, and His) supplemented with sucrose or glucose at indicated concentrations (0.02, 0.04, 0.2, 0.4, or 2.0% with corresponding values at mM indicated below the percentages), and the β-gal activities were measured with these yeast cells. Each value is the mean ± SE of five independent biological determinations. The values of β-gal activity in different sucrose concentrations were statistically compared, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test). Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Sucrose, like Glucose, Inhibits Seed Germination. The wild-type Col-0 seeds were planted in the sugar-free medium (indicated by asterisk) or the medium supplemented with 0.5% (equivalent to 15 mM sucrose, 28 mM glucose or mannitol or sorbitol), 1% (equivalent to 29 mM sucrose, 56 mM glucose, or 55 mM mannitol or sorbitol), or 3% (equivalent to 88 mM sucrose, 167 mM glucose, or 165 mM mannitol or sorbitol) sucrose, glucose, mannitol, or sorbitol, and germination rate was recorded 16 h (top) and 24 h (bottom) after stratification. Each value is the mean ± SE of five independent biological determinations, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group of values. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Down-Regulation of SUT4 and the Cytochrome b5 Cyb5-2 Confers Sucrose/Glucose-Insensitive Phenotypes in Sucrose/Glucose-Induced Inhibition of Seed Germination. The sut4 and cyb5-2 single mutants and sut4 cyb5-2 double mutants show marked sucrose-insensitive phenotypes in sucrose-induced inhibition of seed germination, and slight glucose-insensitive phenotypes in glucose-induced inhibition of seed germination. Seeds were planted in the sugar-free medium (0) or the media supplemented with 0.5, 1, or 3% sucrose or glucose or mannitol, and germination rate was recorded 16 and 24 h after stratification. Seed germination was also tested in a complementation line for the cyb5-2 mutant (cyb5-2/Cyb5-2) or for the sut4 mutant (sut4/SUT4). Col, wild-type Col seeds. Each value is the mean ± SE of five independent biological determinations, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group of values. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Two Other Members of Cytochrome b5, Cyb5-4 and Cyb5-6, but Not Cyb5-1 or SUT1, Are Required for the Response of Seed Germination to Sucrose and Glucose. (A) The expression levels of the Cyb5-1 (i), Cyb5-4 (ii), and Cyb5-6 (iii) genes, respectively, in the cyb5-1, cyb5-4, and cyb5-6 mutants, which shows the three mutants are knockdown alleles. Each value is the mean ± SE of three independent biological determinations. RT–PCR analysis of SUT1 expression in wild-type Col and homozygous sut1 mutant shows that the sut1 mutant is a knockout allele (iv) where Actin served as a control. (B) The marked sucrose-insensitive phenotypes in sucrose-induced inhibition of seed germination and slight glucose-insensitive phenotypes in glucose-induced inhibition of seed germination were observed in the cyb5-4 and cyb5-6 mutants, but not in cyb5-1 and sut1 mutants. Col, wild-type Col seeds. Each value is the mean ± SE of five independent biological determinations, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group of values. (C, D) Expression of Cyb5-4 and Cyb5-6 rescues the cyb5-4 and cyb5-6 mutant phenotypes. (C) Real-time PCR analysis shows that the Cyb5-4 and Cyb5-6 genes were overexpressed, respectively, in the complemented lines of cyb5-4 (cyb5-4/Cyb5-4, left panel) and cyb5-6 (cyb5-6/Cyb5-6, right panel) mutants. (D) The complemented lines of cyb5-4 (cyb5-4/Cyb5-4) and cyb5-6 (cyb5-6/Cyb5-6) mutants display wild-type phenotypes in the sucrose-induced inhibition of seed germination, which was tested in the media containing 0, 0.5, or 3% sucrose (Suc) or 0.5% glucose (Gluc) or 0.5% mannitol (Man) 16 and 24 h after stratification. Col, wild-type Col seeds. Each value in (B) and (D) is the mean ± SE of three independent biological determinations, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group of values. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 ABA, But Not ABI2/4/5-Mediated Signaling, Is Required for the Response to Sucrose, while Exogenous ABA Treatment Does Not Change the Sucrose Insensitivity of the sut4 and cyb5-2 Mutants in Seed Germination. (A) Germination of the aba2 mutant seeds in the sugar-free medium (0) and the media containing different concentrations of sucrose or glucose (as indicated). The germination rates were recorded 18 h (top), 24 h (middle), and 36 h (bottom) after stratification. (B) Germination of the abi2, abi4, and abi5 mutant seeds in the sugar-free medium (0) and the media containing different concentrations of sucrose or glucose (as indicated). The germination rates were recorded 18 h (top), 24 h (middle), and 36 h (bottom) after stratification. (C) Germination of the sut4 and cyb5-2 mutant seeds in the media containing 0.5% sucrose and supplemented with 0, 0.5, or 1 μM (±)-ABA. The germination rates were recorded 16 h (top), 24 h (middle), and 36 h (bottom) after stratification. (D) Germination of the sut4 and cyb5-2 mutant seeds in the media containing 3% sucrose and supplemented with 0, 0.2, 0.5, 1, or 3 μM (±)-ABA. The germination rates were recorded 24 h (top), 36 h (middle), and 48 h (bottom) after stratification. In (A)–(D), Col, wild-type Col seeds. Each value is the mean ± SE of five independent biological determinations, and different letters indicate significant differences at P < 0.05 (Duncan’s multiple range test) when comparing values within the same group of values. Molecular Plant 2012 5, 1029-1041DOI: (10.1093/mp/sss001) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions