1. Brassinosteroids Regulate the Differential Growth of Arabidopsis Hypocotyls through Auxin Signaling Components IAA19 and ARF7 
Xiao-Yi Zhou, Li Song, Hong-Wei Xue 
Molecular Plant 
Volume 6, Issue 3, Pages (May 2013)
DOI: /mp/sss123
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Effects of BRs on Dark-Grown Arabidopsis Seedlings.
(A) Deficient or excessive BR results in abnormal morphology of etiolated seedlings. Six-day-old seedlings were grown on MS medium in the absence or presence of BL (100nM) under 16-h light/8-h dark cycles or continuous dark. Bar = 1 cm.
(B) Etiolated seedlings exhibit morphological changes under BR treatment, including hypocotyl curvatures and elongation inhibition, which are enhanced by bzr1-D mutation. Six-day-old seedlings were grown on MS medium containing different concentrations of BL in darkness (upper panel) and hypocotyl length was calculated and shown as the average ± SE (n > 20, bottom panel). Statistical analysis was performed using a two-tailed Student’s t-test (** P < 0.01, compared with WT). Bar = 1 cm.
(C) Observation of the BR-caused curvatures indicates that cells at the convex sides are longer than those at the concave sides. Six-day-old dark-grown seedlings under mock (upper panel) or BL treatment (100nM, bottom panel) were stained with propidium iodide. The cell wall verges of star-marked cells were highlighted by arrows. Bar = 100 μm.
(D) Hypocotyl curvatures of 4-day-old dark-grown seedlings after 90° reorientation for 24 h (upper panel, bar = 1 cm). The time-course measurements showed that BR enhances the gravitropic response (bottom panel). Data are presented as mean ± SE (n > 60) with three independent replicates. Statistical analysis was performed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT).
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Effect of BR on Auxin Distribution in Etiolated Hypocotyl.
(A) Auxin distribution by observing DR5::GUS expression in the etiolated seedlings treated with BL (50 nM), BL and 2,4-D, or BL and NPA. Six-day-old dark-grown seedlings were used for staining. Bar = 2 mm.
(B) Enlarged pictures shows that both uniform (1, 2, 5, 9, 10, 11) and asymmetric (3, 4, 6, 7, 8) stainings can be observed at the curved (2, 3, 5, 6, 7, 9) or straight (1, 4, 8, 10, 11) regions. Arrows highlight the regions accumulated with staining signals. Time upon the panels indicate staining time. Bar = 500 μm.
(C) Calculation of the BR-caused curvatures and auxin distribution patterns. Proportion of different auxin distribution patterns at the curvatures of BL-treated hypocotyls was calculated and was presented as mean ± SE (n > 20). Asymmetric, DR5::GUS signal is accumulated at the convex side; Reverse, DR5::GUS signal is accumulated at the convave side; NS, no detecting signals. The experiments were repeated three times.
(D) Etiolated hypocotyl morphology under BL (50nM) combined with 2,4-D treatment (left panel) or 2,4-D alone treatment (right panel). Bar = 1 cm. (E) Etiolated hypocotyl morphology under BL (50nM) combined with NPA treatment (left panel) or NPA alone treatment (right panel). Bar = 1 cm.
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Mutations of IAA19 and ARF7 Altered BR Sensitivity in the Dark.
(A) Different growth of msg2 seedlings compared with WT. Six-day-old seedlings of WT (upper) and msg2 (bottom) seedlings were grown vertically on MS medium containing different concentrations of BL in darkness. Bar = 1 cm.
(B) Relative hypocotyl length of 6-day-old seedlings of WT, tir1, axr1, axr2, and msg2 under dark (left panel) or continuous white light (~60 μmol m–2 s–1, right panel) grown on MS medium containing different concentrations of BL. Length of hypocotyls in the mock treatment was set as 1. Error bars indicate SE (n > 20) and statistical analysis was performed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT).
(C) Growth of 6-day-old seedlings of WT and arf7 on MS medium in the absence or presence of BL (100nM) in darkness (left panel, bar = 1 cm). Relative length of hypocotyls was calculated (hypocotyl length in the mock treatment was set as 1) and statistical analysis was performed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT, right panel). Error bars indicate SE (n > 20).
(D) Six-day-old seedlings of WT, msg2, and different arfs grown vertically on MS medium in the absence (upper panel) or presence of BL (100nM, bottom panel) in darkness. Bar = 1 cm.
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 msg2 or arf7 Are Epistatic to bzr1-D.
(A) Yeast two-hybrid analysis revealed that there is no interaction between BIN2 and ARF7 or IAA19. The known IAA19–ARF7 interaction was used as a positive control. The empty vectors AD (pGADT7) and BD (pGBKT7) were used as the negative control. Transformed yeast cells were grown on SD/-Trp/-Leu/-His/-Ade (-His -Ade) or SD/-Trp/-Leu (+His +Ade) medium.
(B) qRT–PCR analyses revealed the altered expressions of IAA19 and ARF7 under BR treatment or in bzr1-D. WT seedlings were grown on MS medium containing 100nM BL for 6 d or treated with BL (100nM) for 3 or 6 h. bzr1-D seedlings were grown in darkness for 7 d. Relative transcription was calculated by setting the expression of WT under mock treatment as 1. AtACTIN2 was used as internal control and data are presented as mean ± SD (n = 3). Experiments were repeated three times, with similar results.
(C) bzr1-D msg2 and bzr1-D arf7 suppressed the enhanced hypocotyl curvature and elongation inhibition of bzr1-D under BL treatment (100nM, upper panel, bar = 1 cm). Seedlings were grown vertically on MS medium containing different concentrations of BL in darkness for 6 d and data were presented as mean ± SE (n > 30, bottom panel). Statistical analysis was performed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with bzr1-D).
(D) bzr1-D msg2 and bzr1-D arf7 showed restored phenotype of enhanced gravitropic response of bzr1-D. Seedlings were grown vertically on MS medium in dark for 4 d and observed after 90° reorientation for 24 h (upper panel, bar = 1 cm). The gravitropic curvature angles were quantified time-coursely and data are presented as mean ± SE (n > 50) of three independent repeats. Statistical analysis was performed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with bzr1-D).
(E) msg2 or arf7 mutation partially restores the phenotype of bzr1-D in light. The bzr1-D msg2 and bzr1-D arf7 show partially recovered phenotypes of bzr1-D, including the shape of rosette leaves and the length of petioles. Plants were germinated on MS medium for 6 d and then transplanted to soil for 14 d. One of the 4th pair’s rosette leaves are shown (right panel). Bar = 1 cm.
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 BZR1 Binds to the Promoter Regions of IAA19 and ARF7.
(A, B) EMSA showed that recombinant BZR1-His protein binds to the IS1 region (–184 to –6) of IAA19 promoter (A), and regions AS1 (–193 to 0), AS2 (–393 to –146), and AS3 (–277 to –63), but not AS4 (–1890 to –1691), of ARF7 promoter (B). The unlabeled DNA (~20 to ~120-fold in concentration) was used as the competitor for assays. Recombinant CRC-His protein was used as the negative control. Arrows highlight the shift bands. The schematic of the promoter regions and locations of the BRRE and E-box elements are indicated.
(C) ChIP analysis confirmed that BZR1 binds to IAA19 and ARF7 promoter regions. Six-day-old etiolated seedlings of pBZR1::mBZR1–CFP transgenic plants and WT were used and the precipitated DNA was quantified by qRT–PCR. Enrichment fold was calculated as the ratio between transgenic and WT seedlings by normalizing to that of the RPS16A coding region. The CPD promoter region was used as a positive control. The positions of the ChIP amplicons for IAA19 promoter (IC1) and ARF7 promoter (AC1) were indicated in (A) or (B). Data are presented as mean ± SD of three replicates. Similar results were obtained in three independent experiments.
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
BR Effects on Global Gene Expression Are Reduced in msg2 and axr2.
(A) M–A plots showed the changed BR-regulated gene expression. Each plot corresponds to a transcript detected by one probe. M = log2(I/C), where I represents the gene expression value in BL-treated samples (WT-BL, msg2-BL, or axr2-BL) and C represents that in control samples (WT, msg2, or axr2). The genes induced under BL treatment (M > 1) are in red, while repressed (M < –1) are in green.
(B) Summary of the differentially expressed genes (fold change > 2 and FDR-correlated P-value < 0.1). ‘WT-BR vs WT’, ‘msg2-BR vs msg2’, and ‘axr2-BR vs axr2’ indicate the BR-regulated genes in WT, msg2, and axr2, respectively. ‘msg2 vs WT’ and ‘axr2 vs WT’ indicate the altered genes in msg2 or axr2 seedlings versus WT.
(C) Hierarchical clustering of the BL-regulated genes in WT and msg2. The bottom bar shows the color scale for regulation ratio (log2) relative to the control samples.
(D) Venn diagram showing the number of overlapping or unique BR-regulated genes in WT and msg2 seedlings.
(E, F) Significantly over-represented biological process terms according to Gene Ontology categories for 766 genes responding to BR in WT but not in msg2 (E) and 969 genes affected by msg2 mutation (F). Hypergeometric test was used to examine the significance of the over-representation and the length of bars represents negative logarithm (base 10) of the adjusted P-value.
(G) Venn diagram showing the number of overlapping or unique BR-regulated genes in WT, msg2, and axr2 seedlings. The dotted panes represent the genes with impaired BR response in msg2 specifically (listed in Table 2).
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Expression Patterns of BR-Regulated Genes and Hypothetical Model of Effects of IAA19 and ARF7 in Mediating the BR Effect on Hypocotyl Differential Growth in Dark.
(A) qRT–PCR analysis confirmed the expression patterns of SIBS genes (At1g26380, At5g05340, At4g37160, At5g47980, At5g12380, and At1g25250) and genes with impaired BR response in msg2 (At1g78990, At5g04310, At2g19150, and At3g29670). Four-day-old seedlings of WT, axr2, msg2, and arf7 grown in dark were used. Gene expression level under BL treatment (100nM) versus mock treatment are presented as mean ± SD (n = 3). Experiments were repeated three times, with similar results. AtACTIN2 was used as the internal control.
(B) BR activates the dual function transcription factor BZR1, which represses the expression of IAA19, a repressor of ARF7, and stimulates the ARF7 transcription through direct binding to their promoter regions. These regulations result in the accumulation of activated ARF7 protein and transcriptional changes of downstream genes, leading to the differential growth and hence the morphogenesis of dark-grown seedlings. Auxin also regulates the differential growth through IAA19 and ARF7, of which ARF7 mediates the inducement of IAA19 expression, generating a feedback loop to precisely control the differential growth.
Molecular Plant 2013 6, DOI: ( /mp/sss123)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions