A Dual-Function Transcription Factor, AtYY1, Is a Novel Negative Regulator of the Arabidopsis ABA Response Network  Tian Li, Xiu-Yun Wu, Hui Li, Jian-Hui Song, Jin-Yuan Liu  Molecular Plant  Volume 9, Issue 5, Pages 650-661 (May 2016) DOI: 10.1016/j.molp.2016.02.010 Copyright © 2016 The Author Terms and Conditions

Figure 1 AtYY1 Is a Nuclear-Localized Protein with Both Repression and Activation Domains. (A) Genomic structure of the AtYY1 (At4g06634.1) gene. Exons are shown as boxes and introns are shown as lines. The triangles indicate the position of the yy1 mutant T-DNA insertion. The predicted AtYY1 protein is depicted below. Zinc-finger domains (Zn Finger), putative nuclear localization signal (NLS), and the acidic region are shown as boxes in purple, blue, and yellow, respectively. (B) AtYY1 subcellular localization. GFP and AtYY1-GFP fusions under the control of the CaMV 35S promoter were transiently expressed in onion epidermal cells. (C) AtYY1 functional domains were mapped using transient expression assays. The GAL4-LUC reporter and GAL4DB fusion effector plasmids were co-bombarded in tobacco leaves. (D) Identification of the AtYY1 repression domain using active repression assays. AtDREB1A-activated LUC activity was differentially affected by co-bombardment with the GAL4DB fusion effector plasmids. 35S-NOS blank plasmid (Null) and 35S-AtDREB1A-NOS effector plasmid were used as negative and positive controls, respectively. The data were derived from three independent experiments; error bars indicate the standard deviation. Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 2 AtYY1 Tissue and Induction Expression Profiles. (A) Tissue and developmental expression of AtYY1 in Arabidopsis by northern blot analysis. Total RNA was isolated from Arabidopsis mature seeds (Sd), seedlings (Sl), roots (R), stems (S), leaves (L), flowers (F), and pods (P) at the indicated developmental stages. The rRNA bands in ethidium bromide-stained gels are shown as loading controls. (B) Semi-quantitative RT–PCR analysis of AtYY1 expression. Total RNA was isolated from seed sprouts (Sp), roots (R), aerial seedling parts (AP), stems (S), young rosette leaves (YRL), mature rosette leaves (MRL), old rosette leaves (ORL), mature stem leaves (MSL), flowers (F), upper part of stems (YS), middle and lower stem parts (OS), and seeds (Sd). Actin2 was used as a control to show the normalization of the template amount in PCR reactions. (C) Histochemical localization of GUS activity in pAtYY1::GUS transgenic Arabidopsis plants. (a–d) Seeds that germinated for 36 h, 48 h, 60 h, and 72 h; (e–h) 5-, 8-, 15-, and 25-day-old seedlings; (i–k) 20-, 30-, and 45-day-old stem leaves; (l–n) 20-, 30-, and 45-day-old rosette leaves; (o) flower; (p) sepal; (q) petal; (r) silique and seeds. Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 3 AtYY1 Represses the Plant ABA and Salt Response. (A) RT–PCR analysis of AtYY1 expression in wild-type (WT), yy1 mutant, and four independent 35S::HA-AtYY1 transgenic lines. (B) Verification of HA-AtYY1 fusion protein expression in the 35S::HA-AtYY1 transgenic lines by immunoblot analysis. Coomassie staining of the RuBisCO large subunit (RbcL) is shown as loading control. (C) Quantitative RT–PCR analysis of AtYY1 expression under various treatments. 10-day-old seedlings were treated with 100 μM ABA, 300 mM NaCl, 350 mM mannitol, or dehydration for the indicated times. (D and E) Germination rates of WT, yy1, and 35S::HA-AtYY1 transgenic seeds incubated on MS medium containing 0, 0.3, or 0.5 μM ABA; and 0, 150, or 200 mM NaCl for 2 days; *P < 0.05 and **P < 0.01 (t-test). (F) Comparison of relative root growth among WT, yy1, and transgenic plants. Three-day-old seedlings grown on MS medium were transferred to MS medium containing 0, 1.0 μM ABA, or 150 mM NaCl, and grown vertically for 5 days. Scale bars represent 1 cm. (G) Comparison of cotyledon greening ratios of WT, yy1, and transgenic plants. Seeds geminated on MS medium for 2 days were transferred to MS medium containing 0, 0.5 μM ABA, or 150 mM NaCl, and grown for 5 days. Green cotyledon percentages are indicated below each panel. Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 4 AtYY1 Impairs Arabidopsis Dehydration Resistance. (A) ABA-induced stomatal closure in wild-type (WT), yy1 mutant, and AtYY1-overexpressing plants (OE1). Stomata were observed in the epidermal guard cells treated with either 0 or 25 μM ABA. (B) Relative stomatal apertures compared with those grown on ABA-free medium. The results are from three replicates (n = 30); *P < 0.05 and **P < 0.01 (t-test). (C) Measurement of water loss rates for WT, yy1 mutant, and AtYY1-overexpressing plants. (D) Dehydration tolerance phenotypes of WT, yy1 mutant, and AtYY1-overexpressing plants. 2-week-old Arabidopsis seedlings were dehydrated on dry filter paper in Petri dishes for 12 h and then re-watered for 36 h. Survival rates were determined for at least 100 plants per line. (E) Drought tolerance phenotypes of WT, yy1 mutant, and AtYY1-overexpressing plants. 3-week-old Arabidopsis plants were not watered for 20 days and then re-watered for 3 days. Survival rates are shown on the right. Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 5 AtYY1 Directly Regulates ABR1. (A) Microarray analysis of representative genes demonstrates dose-dependent expression of AtYY1. (B) AtYY1 can bind an ABR1 promoter sequence containing a conserved YY1 binding site (CCATATT) in vitro. The sequences of the oligonucleotide (P) or mutant probes (M1–M4) used are listed above. The YY1 binding motif is underlined and mutant sites are shown in red. Biotin-labeled probes (100 fmol) were incubated with His-AtYY1 protein (3 μg) or His-tag alone. Varying concentrations of unlabeled probe (10- to 1000-fold) or unlabeled mutant probes (1000-fold) were used for the competition assays. (C) Transcriptional activity of AtYY1 on ABR1 promoter YY1 binding sites. The LUC reporters were driven by three tandem wild-type or mutant YY1 binding sites (3× YY1 or 3× mYY1). The sequences of YY1 and mYY1 binding sites were the same as P and M1 in (B), respectively. GAL4DB (GB) effector plasmid was used as negative control; **P < 0.01 (t-test). (D) ChIP–qPCR analysis of AtYY1 binding to the ABR1 promoter in vivo. ABR1 structure and the positions of the qRT–PCR primers corresponding to the promoter (F1 and R1) and the coding region (F2 and R2) are indicated. The blue circle indicates the YY1 binding site. An Actin2 gene fragment was used as the negative control; **P < 0.01 (t-test). (E and F) Comparison of ABR1 and ABA-responsive gene expression levels between WT and yy1 seedlings in response to treatment with 100 μM ABA (E) and 300 mM NaCl (F). Relative expression levels were determined by qRT–PCR using Actin2 as an internal standard. Data are presented as the mean ± SD (n = 3); *P < 0.05 and **P < 0.01 (t-test). Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 6 ABI4 Positively Regulates AtYY1 and this Regulation Can Be Antagonized by ABR1. (A) Comparison of AtYY1 expression patterns among wild-type, abi1-1, abi4-1, and abr1-3 mutants, and of ABI1, ABI2, and ABI4 expression in wild-type and yy1 mutants. 10-day-old wild-type and mutant seedlings were treated with 100 μM ABA for the indicated time periods; *P < 0.05 and **P < 0.01 (t-test). (B) ABR1 antagonizes ABI4 by competing for CE1 site binding within the ABI5 and AtYY1 promoters. The LUC reporter was driven by three tandem CE1 sites (3× CE1) from the ABI5 or AtYY1 promoters. 35S-NOS blank effector plasmid (Null) was used as negative control; **P < 0.01 (t-test). Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions

Figure 7 A Working Model for AtYY1 Functions in ABA Response. The arrows indicate positive regulation and the nail shapes indicate negative regulation. Molecular Plant 2016 9, 650-661DOI: (10.1016/j.molp.2016.02.010) Copyright © 2016 The Author Terms and Conditions