1. Volume 9, Issue 6, Pages 870-884 (June 2016)
Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue 
Shin Ae Lee, Sejeong Jang, Eun Kyung Yoon, Jung-Ok Heo, Kwang Suk Chang, Ji Won Choi, Souvik Dhar, Gyuree Kim, Jeong-Eun Choe, Jae Bok Heo, Chian Kwon, Jae-Heung Ko, Yong-Sic Hwang, Jun Lim 
Molecular Plant 
Volume 9, Issue 6, Pages (June 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1 Regulation of GAZ mRNA Abundance by GA and ABA.
(A) Expression of GAZ in 7-day-old wild-type (WT), ga1-3, ga1-3 plus 10 μM GA3, and scl3-1 ga1-3 roots. The value for WT is set to 1 and the values relative to WT are shown.
(B) GAZ mRNA abundance in the loss (scl3-1) and gain (SCL3-OX) of SCL3 function. The upper panel shows a heatmap of the GAZ expression levels from ATH1 microarray experiments using scl3-1 and SCL3-OX roots. The lower panel illustrates qRT–PCR results of GAZ transcript levels in scl3-1 and SCL3-OX. The value for WT is set to 0, and expression values relative to WT are shown as numerical values for log2-fold change.
(C) qRT–PCR of the GAZ mRNA level in 7-day-old WT roots in the absence or presence of ABA (10 μM).
(D) The GAZ transcript levels in 7-day-old WT, XER-OX, and aba2-2 roots.
(E) Transient expression assay of GAZ promoter activity in the absence or presence of ABA (10 μM). The value with no ABA is set to 1, and the relative value of luciferase (LUC) activity with ABA is shown.
Error bars indicate mean ± SE from three independent replicates. Statistically significant differences were determined by Student's t-test (*p < 0.05; **p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 Transcriptional Activity of GAZ and Its Nuclear Localization.
(A) Schematic representation of the constructs used for transient expression assays. The reporter consists of the 35S promoter (Pro35S), five repeats of the GAL4 binding sequence (5xUAS), a firefly luciferase coding sequence (LUC), and a NOS terminator (NOS). The effector constructs have either GAL4 DNA-binding domain (GAL4DB) only or GAL4DB-GAZ fusion under the control of the 35S promoter.
(B) Arabidopsis protoplast transient expression assays of GAL4DB (GAL4-only) or GAL4DB-GAZ fusion (GAL4-GAZ). The value for GAL4-only is set to 1 as a control, and the value relative to the control is shown. Error bars indicate the mean ± SE from three independent replicates. Significance of difference was statistically determined by Student's t-test (*p < 0.05).
(C) Confocal image of GAZ-GFP localization (Pro35S:GAZ-GFP) in the nuclei of root cells.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 Tissue-Specific Expression of GAZ in the Root.
(A and B) Expression of the ProGAZ:GUS transcriptional fusion in the primary root at 7 dpg (A) and in the transverse section of the root (B).
(C) Expression of the ProGAZ:H2B-YFP transcriptional fusion in the primary root.
(D–D″) Expression of the ProGAZ:GAZ-GFP translational fusion in the primary root. The insets in (D) are shown in (D′) and (D″). Unlike the transcriptional fusions, the localization of GAZ-GFP by the translational fusion is detected ubiquitously in the root, although the intensity of fluorescence is weak.
Epidermis (EP), cortex (CO), endodermis (EN), and stele (ST) are indicated.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 GAZ Plays a Role in the GA-Mediated Control of MC Formation.
(A) Schematic illustration of the Arabidopsis root. Longitudinal view of the root apical meristem at an early stage in post-embryonic root development (left). The CEI in the stem cell niche generates CEI itself and CEID by an anticlinal ACD. Subsequently, the CEID undergoes a periclinal ACD to generate the cortex and the endodermis. Mature ground tissue (GT) later in root development (right). The endodermis undergoes additional periclinal ACDs, resulting in the endodermis (EN; yellow) and the middle cortex (MC; red), which is located between the endodermis and the cortex (CO; light blue). Thus, the mature primary root has three layers in the GT: endodermis (EN), middle cortex (MC), and cortex (CO).
(B) Quantitative evaluation of MC formation in WT, GAZ-OX, and GAZ-SRDX roots under standard, GA (10 μM GA3), and PAC (1 μM) conditions.
(C–E) Confocal images of MC formation in WT (C), GAZ-OX (D), and GAZ-SRDX roots (E) in the presence of PAC. The insets in (C) and (E) illustrate periclinal ACDs that generate the endodermis (EN) and middle cortex (MC) layers. The endodermis (EN), middle cortex (MC), and cortex (CO) layers are indicated with white arrowheads.
(F) Percent of MC formation in WT, scl3-1, GAZ-OX, and scl3-1 GAZ-OX under PAC treatment. The frequency of periclinal ACDs for MC formation was analyzed at 7 dpg.
Error bars indicate the SE from three independent replicates. Significance of difference was statistically determined by Student's t-test (*p < 0.05; **p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5 ABA Plays a Role in MC Formation during GT Maturation.
(A) Quantitative analysis of MC formation in WT seedlings at 7 dpg and 12 dpg in the absence or presence of 1 μM ABA. ABA influences the early phase of GT maturation more profoundly.
(B) Percent of MC formation in WT, aba2-2, xer, and XER-OX at 7 dpg. Error bars represent the SE of three biological replicates. Statistically significant differences were determined by Student's t-test (*p < 0.05; **p < 0.01).
(C–F) Confocal images of WT (C), aba2-2 (D), xer (E), and XER-OX roots (F) at 7 dpg. The insets in (D) and (E) illustrate periclinal ACDs of the endodermis during MC formation. ABA-deficient mutants (aba2-2 and xer) displayed precocious MC formation, whereas MC formation in ABA-overproducing XER-OX seedlings was delayed. The endodermis (EN), middle cortex (MC), and cortex (CO) layers are indicated with white arrowheads. Bar = 50 µm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6 Interplay between ABA and GA in MC Formation.
(A) Quantitative analysis of MC formation with increasing ABA concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 μM) in the absence or presence of 10 μM GA3.
(B) Percent of MC formation in WT, aba2-2, and xer under standard, GA (10 μM GA3), and PAC (1 μM) conditions.
(C) Percent of MC formation in WT and ga1-3 in the absence or presence of ABA (1 μM).
(D) Quantitative evaluation of MC formation in WT, scl3-1, abi4-1, and scl3-1 abi4-1 under ABA treatment.
(E) Confocal image of scl3-1 abi4-1 in the presence of ABA. The inset illustrates periclinal ACDs that generate the endodermis (EN) and middle cortex (MC) layers.
Error bars indicate the SE from three biological replicates. Significance of difference was statistically determined by Student's t-test (*p < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
GAZ Serves as a Convergent Point of ABA and GA during MC Formation.
(A) Percent of MC formation in WT, GAZ-OX, and GAZ-SRDX in the absence or presence of ABA (1 μM). GAZ-OX seedlings were hypersensitive to ABA, whereas GAZ-SRDX seedlings were insensitive to ABA in MC formation.
(B and C) Confocal images of GAZ-OX and GAZ-SRDX roots in the presence of ABA.
(D) Quantitative analysis of MC formation in WT, scl3-1, GAZ-OX, and scl3-1 GAZ-OX under ABA treatment. scl3-1 GAZ-OX showed premature MC formation compared with scl3-1 and GAZ-OX single mutants.
(E) Confocal image of scl3-1 GAZ-OX under ABA treatment.
Error bars represent the SE from three independent biological replicates. Significance of difference was statistically determined by Student's t-test (*p < 0.05; **p < 0.01).
The insets in (C) and (E) illustrate periclinal ACDs for MC formation. The white arrowheads indicate the endodermis (EN), middle cortex (MC), and cortex (CO) layers.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8
GAZ Is Involved in the Transcriptional Regulation of GA Biosynthesis and ABA Catabolism Genes.
(A–E) Expression analyses of GA20ox1 to GA20ox5 in 7-day-old WT and GAZ-OX roots under standard, GA, or PAC conditions.
(F–I) Expression of GA3ox1 to GA3ox4 in 7-day-old WT and GAZ-OX roots under standard, GA, or PAC conditions.
(J–M) qRT–PCR results for CYP707A genes (CYP707A1 to CYP707A4) in 7-day-old WT and GAZ-OX roots in the absence or presence of ABA. GAZ regulates the abundance of CYP707A1 mRNA.
Error bars indicate the SE from three biological replicates. Statistical significance of difference was determined by Student's t-test (*p < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

10. Figure 9
Schematic Diagram of the Hormonal Control of MC Formation during GT Maturation.
The GA signaling pathway, including SCL3, regulates GAZ expression. Regulation of GAZ expression by SCL3 is indirect. Thus, another unknown TF (TF X), which directly regulates transcription of GAZ, should be positioned downstream of SCL3 and upstream of GAZ. In parallel, the ABA pathway also controls GAZ mRNA abundance. GA biosynthesis and ABA catabolism are subject to the feedback regulation, which also involves GAZ in the root. In particular, GAZ regulates the expression levels of GA20ox and GA3ox (GA biosynthesis) and CYP707A1 (ABA catabolism). Thus, GAZ, serving as a convergent point for the interaction between the ABA and GA pathways, plays a role in maintaining a constant flux of ABA and GA, which in turn modulates the timing of MC formation during GT maturation.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions