1. Volume 7, Issue 6, Pages 1006-1025 (June 2014)
Negative Feedback Regulation of Auxin Signaling by ATHB8/ACL5–BUD2 Transcription Module 
Baima Simona , Forte Valentina , Possenti Marco , Peñalosa Andrés , Leoni Guido , Salvi Sergio , Felici Barbara , Ruberti Ida , Morelli Giorgio  
Molecular Plant 
Volume 7, Issue 6, Pages (June 2014)
DOI: /mp/ssu051
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 ACL5 and BUD2 Are Direct Targets of ATHB8.
(A) Schematic representations of the ACL5 and BUD2 genes. Green boxes represent BS-IIIACL5 and BS-IIIBUD2 and their position is indicated. Red and blue boxes correspond to the DNA fragments assayed by ChIP (positive and negative, respectively). Orange regions show 5’UTR, black boxes indicate exonic sequences, whereas light gray indicates intronic ones.
(B) ChIP of ATHB8pro:ATHB8–GFP seedlings using an anti-GFP antibody. The immunoprecipitates were analyzed by the presence of the specific promoter region of ACL5 or BUD2 by qPCR. Fold enrichment of DNA fragments containing BS-IIIACL5 (left) or nearby BS-IIIBUD2 (right) in relation to the total chromatin input, normalized with the control line (ATHB8pro:GFP) is shown in four independent ChIP experiments (I, II, III, and IV). The error bars represent SD (n = 3). Fold enrichments, compared with the controls, are statistically significant (Student’s t-test; p < 0.05).
(C) Electrophoretic Mobility Shift Assay with a DNA fragment of ACL5 promoter bearing the GCAATCATTAC motif (BS-IIIACL5, lanes 1–5) and a derivative containing the GCGCTCAGGAC mutated sequence (mutBS-III ACL5, lanes 6–8). The retardation assays were performed either with the GST protein (lanes 2 and 7) or with GST–HD–Zip-8 protein (lanes 3–5 and 8). An excess of unlabeled competitor DNA was added to the reaction mixtures in lanes 4 and 5.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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3. Figure 2
ACL5 Expression Pattern in Leaf Primordia, Primary Root, and LRP.
(A) Histochemical localization of GUS activity in leaf primordia in 4, 5, 6, and 7 d after germination (DAG) seedlings of ATHB8pro:GUS, and in transgenic lines expressing in Col-0 background ACL5pro:ACL5–GUS (A5w1, A5w2) and ACL5mutpro:ACL5–GUS (A5w2mut, A5w3mut). For each time point, pattern analysis was made in leaf primordia of the same area to ensure comparable developmental stages. The area of the primordia is indicated below the age. The inset shows a detail of the elongated procambial cells. Scale bar = 100 μm.
(B–E) Histochemical localization of GUS activity in primary root, lateral root primordia before and after emergence of 6-day-old transgenic seedlings expressing ACL5pro:ACL5–GUS (A5w1 (B), A5w2 (C)) and ACL5mutpro:ACL5–GUS (A5w2mut (D), A5w3mut (E)). Arrowheads indicate the position of vascular cells precursors. Scale bar = 100 μm.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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4. Figure 3 High Entopic Expression of ACL5 Affects Vein Formation.
(A) Phenotype of acl5-1 mutant (center), acl5-1 complemented with ACL5pro:ACL5–GUS (left) and ACL5mutpro:ACL5–GUS (right).
(B) Venation pattern of the fifth leaf of 3-week-old plants of Col-0, acl5-1, and acl5-1 complemented lines. Panels show dark-field images of cleared leaves. Scale bar = 1mm.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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5. Figure 4 High Levels of ACL5 Activity Impair Xylem Cell Lignification.
(A–R) DIC optics images of cleared leaves showing the morphology of midvein (A–F), third-order vein (G–L), free-ending vein (M–R), from the fifth leaf of 3-week-old plants. Asterisks and arrows indicate not lignified vascular elements in the midvein (C, D), in the third-order vein (I, J), and in free-ending vein (O, P). The transgenic lines A5w1 and A5w3mut express high tgACL5 levels. The transgenic lines A5w6 and A5w5mut express low tgACL5 levels.
(S–X) Transverse sections from the median portion of the fifth leaf stained with toluidine blue. Red arrowheads indicate lignified xylem elements in the midvein. Scale bar = 50 μm in (S, U, V, W, X) and scale bar = 100 μm in (T).
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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6. Figure 5
Co-Regulation of Auxin Genes and the ATHB8/ACL5–BUD2 Transcription Module by ACL5 Level.
(A) Heat-map representation of relative expression, respect to Col-0, in the aerial part of 3-week-old acl5-1 and transgenic plants expressing ACL5pro:ACL5–GUS and ACL5mutpro:ACL5–GUS in Col-0 or acl5-1 background. Due to a negative feedback loop, eACL5 transcript level is up-regulated in acl5-1 and down-regulated in transgenic lines. RE, relative expression; r, Pearson’s correlation coefficient respect to eACL5, r ≥ 0.90 are in bold.
(B) Correlation plot of relative expression level for selected genes (on y-axes) versus TED6 (on x-axes). Only genes with a Pearson’s correlation coefficient r ≥ 0.90 (B) or r < 0.90 (C) were plotted. The data for the acl5-1 and for transgenic lines expressing low or high levels of tgACL5 are indicated by red, light-green, and dark-green circles, respectively.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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7. Figure 6 The Activity of ACL5 Affects Hypocotyl Elongation.
Hypocotyl elongation of Col-0, acl5-1, A5w1 and A5w2 (ACL5pro:ACL5–GUS), A5w2mut, and A5w3mut (ACL5mutpro:ACL5–GUS) seedlings grown for 7 d in light (A) or for 8h in light and then in darkness for 2.5 d (B). Values are mean ± SE (n ≥ 80).
*P < 0.01 in acl5-1 versus all other lines; ^P < 0.01 in A5a1 or A5w2 versus all other lines.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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8. Figure 7
ACL5 Modulates the Expression of Auxin and Xylem Differentiation-Related Genes in Etiolated Seedlings.
RT–qPCR expression analysis of selected auxin and xylem differentiation-related genes in dark-grown acl5-1, A5w1, and A5w3mut 1-week-old seedlings compared with Col-0. mRNA levels were normalized using ACTIN2 as reference gene. Values are mean ± SD of relative quantification (Log2) from three independent experiments, each with three technical replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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9. Figure 8
HD–ZIP III Gene Silencing Negatively Affects Auxin and Xylem Differentiation-Related Gene Expression.
RT–qPCR expression analysis of selected auxin and xylem differentiation-related genes in 1-week-old seedlings of a transgenic line expressing an estradiol-inducible artificial miRNA165. mRNA levels after induction with estradiol were compared with respect to mock control and normalized using ACTIN2 as reference gene. Values are mean ± SD of relative quantification (Log2) from three independent experiments, each with three technical replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
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10. Figure 9 LAX3 and LAX2 Are Direct Targets of REV.
(A, B) ChIP assays with an anti-GFP antibody from chromatin obtained from rev-5 REVpro:REV–GFP 8/10-day-old seedlings. The immunoprecipitates were analyzed by the presence of specific promoter regions of LAX3 (fragments A–C) (A) or LAX2 (fragments A–G) (B) by qPCR. The fold enrichments of all DNA fragments amplified in the respective gene regions, in relation to the total chromatin input, normalized with the control line (Col-0) is shown for four independent ChIP experiments (I, II, III, and IV). The error bars represent SD (n = 3). Fold enrichments statistically significant are labeled with asterisks (Student’s t-test; * p < 0.05; ** p < 0.01). The horizontal lines above the graphics are schematic representations of the LAX3 (A) and LAX2 (B) genes. The position of the amplified fragment is shown (A–C and A–G fragments in LAX3 and LAX2 promoters, respectively). Amplifications of LAX3 fragments were normalized with amplification of a 77-nt fragment starting at position +167 from ATG. In the case of LAX2 fragments, a 56-nt amplicon starting in position was used for normalization. Orange boxes show 5’UTR and 3’UTR, and dark-gray boxes indicate exonic sequences whereas light gray indicates intronic ones.
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

11. Figure 10
Model of a Positive and a Negative Feedback Loop between Auxin Flow and Gene Expression in Xylem Procambial Cells.
Two counteractive regulatory loops are shown. The positive feedback loop consists of auxin flow. Auxin flow induces MP expression, MP induces auxin signaling genes and, in turn, HD–ZIP III genes. Among them, REV positively reinforce the auxin flow by regulating LAX2, LAX3 (this work), YUCCA5, and TAA1 (Brandt et al., 2012). The negative feedback loop consists of an ATHB8/ACL5–BUD2 module tightly controlling thermospermine biosynthesis. Thermospermine acts as a positive regulator of the bHLH SAC51 translation which, in turn, negatively regulates auxin signaling by an unknown mechanism. The symbol represents a rheostat adjusting the rate of xylem differentiation as a function of auxin flow (i.e. auxin signaling).
Molecular Plant 2014 7, DOI: ( /mp/ssu051)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions