MicroRNA171c-Targeted SCL6-II, SCL6-III, and SCL6-IV Genes Regulate Shoot Branching in Arabidopsis  Wang Long , Mai Yan-Xia , Zhang Yan-Chun , Luo Qian , Yang Hong-Quan   Molecular Plant  Volume 3, Issue 5, Pages 794-806 (September 2010) DOI: 10.1093/mp/ssq042 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Transgenic Plants Expressing MIR171c Exhibit a Reduced Shoot Branching Phenotype. (A) Forty-nine-day-old LD-grown transgenic 35Spro–MIR171c lines. In (A)–(I), the bottom numbers 1–4 denote WT, 35Spro–MIR171c#2, 35Spro–MIR171c#3, and 35Spro–MIR171c#4, respectively. Bar = 3 cm. (B–E) RNA gel blot analysis of miR171c, SCL6-II, SCL6-III, and SCL6-IV expression in 7-day-old LD-grown 35Spro–MIR171c seedlings. Arrows and asterisks indicate the full-length and 3' cleaved products of SCL6-II, SCL6-III, and SCL6-IV transcripts, respectively. rRNA was used as a loading control (lower gel). (F–H) Quantification analysis of shoot branching of 35Spro–MIR171c plants. The y-axis values denote the percentage of plants with no (black), one (gray), or more than two (white) rosette (F), cauline (G), and total (H) branches. (I) Quantification analysis of height of 35Spro–MIR171c plants. Asterisks denote significant differences between the indicated genotypes and the WT (Student's t-test, ** P < 0.01); n ≥ 30; error bars indicate ±SD. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 SCL6-II, SCL6-III, and SCL6-IV Are miRNA171c Targets. (A) Alignment of partial mRNA sequences of SCL6-II, SCL6-III, SCL6-IV, and miR171c. Free energies of duplex structures were calculated using the Mfold method (Zuker, 2003). mSCL6-II, mSCL6-III, and mSCL6-IV are modified mRNAs that harbor synonymous nucleotide substitutions in miR171c binding sites. (B) Fifty-three-day-old LD-grown double transgenic lines. Bar = 3 cm. The lower numbers in (C), (D), (E), and (F) denote the plant genotypes shown in (B). (C) Analysis of mSCL6–LUC fusion protein expression in 21-day-old LD-grown double transgenic 35Spro–mSCL6-II–LUC/35Spro–MIR171c#2, 35Spro–mSCL6-III–LUC/35Spro–MIR171c#2, and 35Spro–mSCL6-IV–LUC/35Spro–MIR171c#2 plants. n = 3; error bars indicate ±SD. (D) RNA gel blot analysis of miRNA171c expression in the double transgenic lines. rRNA was used as a loading control (lower gel). (E, F) Quantification analysis of total shoot branches (E) and plant height (F) of transgenic plants. Asterisks denote significant differences between the indicated genotypes and the 35Spro–MIR171c#2 transgenic lines (Student's t-test, * P < 0.05, ** P < 0.01); n ≥ 30; error bars indicate ±SD. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Expression Pattern of MIR171 and, SCL6-II, SCL6-III, and SCL6-IV Genes. (A, D, G, J, M, P) GUS activity in 7-day-old LD-grown seedlings of each genotype. (B, E, H, K, N, Q) GUS activity in 15-day-old LD-grown plants of each genotype. (C, F, I, L, O, R) GUS activity in 30-day-old LD-grown flowers of each genotype. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Characterization of the scl6-II, scl6-III, and scl6-IV Mutants. (A) Schematic diagrams displaying T-DNA insertions in the SCL6-II, SCL6-III, and SCL6-IV loci of the scl6-II (FLAG_239F03), scl6-III (SALK_150134), scl6-IV (CS100299) mutants, respectively. P1–P10 are the primers (see Supplemental Table 1) used for PCR-genotyping. (B) PCR analysis of the T-DNA insertions in the SCL6-II, SCL6-III, and SCL6-IV loci in the scl6-II, scl6-III, and scl6-IV single mutants, scl6-II scl6-III, scl6-II scl6-IV, and scl6-III scl6-IV double mutants, and scl6-II scl6-III scl6-IV triple mutants, respectively. (C) RT–PCR analysis of SCL6-II, SCL6-III, and SCL6-IV expression in 7-day-old LD-grown scl6-II scl6-III scl6-IV triple mutant seedlings. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 SCL6-II, SCL6-III, and SCL6-IV Act Redundantly to Promote Branch Formation. (A) Forty-nine-day-old LD-grown WT (lane 1), scl6-II (lane 2), scl6-III (lane 3), scl6-IV (lane 4), scl6-II scl6-III (lane 5), scl6-II scl6-IV (lane 6), scl6-III scl6-IV (lane 7), scl6-II scl6-III scl6-IV (lane 8), and 35Spro–MIR171#2 (lane 9) plants, respectively. Bar = 3 cm. (B, C) Quantification analysis of total shoot branches (B) and plant height (C) of 44–53-day-old LD-grown plants. The lower numbers in (B) and (C) denote the plant genotypes shown in (A). Asterisks denote significant differences between the indicated genotypes and WT (Student's t-test, ** P < 0.01); n ≥ 30; error bars indicate ±SD. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 SCL6-II, SCL6-III, and SCL6-IV Regulate a Variety of Developmental Processes. (A) Twenty-five-day-old LD-grown WT (lane 1), 35Spro–MIR171c#2 (lane 2), and scl6-II scl6-III scl6-IV triple mutant (lane 3) plants. Bar = 3 cm. (B) Leaves from 25-day-old LD-grown WT (lane 1), 35Spro–MIR171c#2 (lane 2), and scl6-II scl6-III scl6-IV triple mutant (lane 3) plants. Bar = 1 cm. (C) Cotyledons from 7-day-old LD-grown WT (lane 1), 35Spro–MIR171c#2 (lane 2), and the scl6-II scl6-III scl6-IV triple mutant (lane 3) seedlings. Bar = 5 mm. (D) Ten-day-old LD-grown WT (lane 1), 35Spro–MIR171c#2 (lane 2), and scl6-II scl6-III scl6-IV triple mutant (lane 3) seedlings. Bar = 1 cm. (E–H) Shoot stem segments of 35-day-old LD-grown WT and scl6-II scl6-III scl6-IV mutant plants showing cauline leaf buds. Arrows indicate abnormal cauline leaf buds. Bar = 1 cm. (I, J) Shoot stem segments of 35-day-old LD-grown scl6-II scl6-III scl6-IV mutant plants showing abnormal leaf patterning. Bar = 1 cm. (K) Flowers from 28-day-old LD-grown WT and scl6-II scl6-III scl6-IV mutant plants. Bar = 1 mm. (L) Measurements of primary root length of 10-day-old LD-grown WT (lane 1), 35Spro–MIR171c#2 (lane 2), and scl6-II scl6-III scl6-IV mutant (lane 3) seedlings. Asterisks denote significant differences between the indicated genotypes and the WT (Student's t-test, ** P < 0.01); n ≥ 30; error bars indicate ±SD. (M, N) Measurements of chlorophyll a and b content in 10-day-old (M) and 25-day-old (N) LD-grown WT (white), 35Spro–MIR171c#2 (black), and scl6-II scl6-III scl6-IV mutant (gray) plants. Asterisks denote significant differences between the indicated genotypes and WT (Student's t-test, * P < 0.05, ** P < 0.01); n = 5; error bars indicate ±SD. Data are representative of three independent datasets, all showing the same effect. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 SCL6-II, SCL6-III, and SCL6-IV Are Localized to the Nucleus. (A) Amino acid sequence alignment of partial SCL6-II, SCL6-III, and SCL6-IV polypeptides showing the putative NLS sequences, which are indicated by asterisks. (B–G) Confocal images of root cells of 5-day-old LD-grown seedlings of transgenic plants expressing GFP (B), GFP–SCL6-II (C), GFP–SCL6-III (D), GFP–SCL6-IV (E), GFP–SCL6-IIΔ343–353 (F), and GFP–SCL6-IVΔ270–280 (G). The left panel indicates images from the GFP channel, and the right panel indicates the images from GFP channel merged with those from the differential interference contrast channel (DIC). Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 8 SCL6-II, SCL6-III, and SCL6-IV Have Transcriptional Activation Activity in Yeast Cells. (A) Bait constructs expressing GUS, CRY1 N-terminal domain (CNT1), SCL6-II, SCL6-III, and SCL6-IV. All proteins are fusions containing the LexA DNA binding domain (LexA). (B) Yeast one-hybrid assay showing that SCL6-II, SCL6-III and SCL6-IV have transcriptional activation activity. More than 12 clones for each construct were taken randomly and analyzed to generate the data. Error bars indicate ±SD. Data are representative of three independent datasets, all showing the same effect. (C) Plate assays showing the transcriptional activation activity. Blue precipitate represents cumulative β-galactosidase activity resulting from activation of the lacZ reporter gene by the SCL6-II, SCL6-III, and SCL6-IV proteins. Quadruplicate yeast patches expressing the indicted LexA hybrid were derived from four independent transformants. At least three independent experiments were performed, and the result of one representative experiment is shown. Molecular Plant 2010 3, 794-806DOI: (10.1093/mp/ssq042) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions