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The Arabidopsis Homeodomain-leucine Zipper II gene family: diversity and redundancy

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Abstract

The Arabidopsis genome contains 10 genes belonging to the HD-Zip II family including ATHB2 and HAT2. Previous work has shown that ATHB2 is rapidly and strongly induced by light quality changes that provoke the shade avoidance response whereas HAT2 expression responds to auxin. Here, we present a genome-wide analysis of the HD-Zip II family. Phylogeny reconstruction revealed that almost all of the HD-Zip II genes can be subdivided into 4 clades (α–δ), each clade comprising 2–3 paralogs. Gene expression studies demonstrated that all the γ and δ genes are regulated by light quality changes. Kinetics of induction, low R/FR/high R/FR reversibility and auxin response analyses strongly suggested that HAT1, HAT3 and ATHB4, as ATHB2, are under the control of the phytochrome system whereas HAT2 is up-regulated by low R/FR as a consequence of the induction of the auxin signaling pathway provoked by FR-rich light. Root and shoot digital in situ revealed that γ and δ genes are also tightly regulated during plant development with both distinct and overlapping patterns. Phenotypes of gain of function and dominant negative lines demonstrated that one or more of the HD-Zip II γ genes negatively regulate cell proliferation during leaf development in a high R/FR light environment. Finally, target gene analysis using a chimeric transcription factor (HD-Zip2-V-G), known to activate ATHB2 target genes in a glucocorticoid-dependent manner, revealed that all the 10 HD-Zip II genes can be recognized by the HD-Zip 2 domain in vivo, implying an intricate negative feedback network.

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Abbreviations

BS1:

Binding site 1

BS2:

Binding site 2

CHX:

Cycloheximide

DEX:

Dexamethasone

DIC:

Differential Interference Contrast

DMSO:

Dimethyl sulfoxide

HD-Zip:

Homeodomain-Leucine Zipper

IAA:

Indole Acetic Acid

MS:

Murashige and Skoog medium

ORF:

Open Reading Frame

qRT-PCR:

quantitative Reverse Transcription-Polymerase Chain Reaction

RT-PCR:

Reverse Transcription-Polymerase Chain Reaction

R/FR:

Red/Far-Red

UPL:

Universal Probe Library

UTR:

Untranslated Region

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Acknowledgements

We thank Bernd Weisshaar for the pBENDER vector. We also thank Takashi Aoyama and Massimiliano Sassi for helpful discussion, and Daniela Bongiorno for skillful technical assistance. This work was supported, in part, by grants from the EU 5th framework project REGIA (QLG2-1999-00876), MUR Strategic Program FIRB 2003 and FIRB ERA-PG, and ASI Biotechnology Program.

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Authors and Affiliations

  1. Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy

    Angela Raffaella Ciarbelli, Andrea Ciolfi, Samanta Salvucci, Valentino Ruzza, Monica Carabelli, Alberto Fruscalzo, Giovanna Sessa & Ida Ruberti

  2. National Research Institute for Food and Nutrition, Rome, Italy

    Marco Possenti & Giorgio Morelli

Authors
  1. Angela Raffaella Ciarbelli
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  2. Andrea Ciolfi
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  3. Samanta Salvucci
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  4. Valentino Ruzza
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  5. Marco Possenti
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  6. Monica Carabelli
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  7. Alberto Fruscalzo
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  8. Giovanna Sessa
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  9. Giorgio Morelli
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  10. Ida Ruberti
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Corresponding author

Correspondence to Ida Ruberti.

Additional information

A. R. Ciarbelli and A. Ciolfi are contributed equally to this work.

Electronic supplemental material

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Supplementary Fig. 1

Alignment of the N-terminal regions of the 10 HD-Zip II proteins. For each column in the alignment, residues conserved in more than 40% of all sequences are highlighted in different colors using BLOSUM62 scores. Blue = K, R; Cyan = F, W, Y; Dark Green = I, L, M, V; Dark Grey = N, S, T; Green = A; Grey = Q; Magenta = H; Purple = P; Red = D, E; Yellow = G (TIFF 2296 kb)

Supplementary Fig. 2

The upstream regions of the HD-Zip II genes are significantly enriched for BS1 and BS2 sequences. S = G or C; W = A or T; N = A, T, C or G. Promoters refer to the 3000 bp-long upstream regions analyzed. The distance between the HD-Zip I genes and the next upstream ORFs is more than 3000 bp in 11 out of 17 genes. For the remaining HD-Zip I genes, the distance is as follows: ATHB5 = 2554 bp, ATHB6 = 1079 bp, ATHB20 = 260 bp, ATHB23 = 1595 bp, ATHB52 = 1267 bp, ATHB54 = 1847 bp. a = Total number of sites identified; b = Expected number of sites based on 1000 randomly sampled groups of promoters; c = Standard deviation of Expected Number; d = Number of promoters containing at least one site; e = Percentage of promoters containing at least one site. >, value is greater than 0.25 (TIFF 521 kb)

Supplementary Fig. 3

HD-Zip II γ and δ genes are induced by low R/FR light. Northern analysis of all the HD-Zip II genes with the exception of ATHB18 in Col-0 seedlings grown in high R/FR (0) and then exposed to low R/FR for the indicated times. Total RNA (A) was used for the analysis of HAT22, HAT1, HAT2, ATHB2, HAT3 transcripts and polyA+ RNA (B) for that of ATHB17, HAT14, HAT9, ATHB4 mRNA expression. ATL18 was used to monitor equal loading (TIFF 3522 kb)

Supplementary Fig. 4

qRT-PCR analysis of ATHB18 in Col-0 seedlings grown in high R/FR (0) and then exposed to low R/FR for the indicated times. The histogram shows the relative expression levels of ATHB18 in high and low R/FR light. Each value is the mean of three separate quantitative PCR reactions normalized to actin 2. Relative transcript abundance of ATHB18 at each time point is normalized to its relative level in Col-0 seedlings in high R/FR (0) (TIFF 1690 kb)

Supplementary Fig. 5

Kinetic of induction of HAT2, IAA19 and IAA29 by low R/FR light. qRT-PCR analysis of HAT2, IAA19 and IAA29 in Col-0 seedlings grown in high R/FR (0) and then exposed to low R/FR for the indicated times. The histograms show the relative expression levels of HAT2, IAA19 and IAA29 in high and low R/FR light. Each value is the mean of three separate quantitative PCR reactions normalized to actin 2. Relative transcript abundance of HAT2, IAA19 and IAA29 at each time point is normalized to their relative levels in Col-0 seedlings in high R/FR (0) (TIFF 1963 kb)

Supplementary Fig. 6

HAT2, but not other light-regulated HD-Zip II genes, is induced by auxin. qRT-PCR analysis of HD-Zip II γ and δ genes in Col-0 seedlings untreated (DMSO) or treated for the indicated times with IAA (1 μM). The graphs show the relative expression levels of ATHB2, HAT1, HAT2, HAT3 and ATHB4 in untreated and IAA-treated seedlings. Each value is the mean of three separate quantitative PCR reactions normalized to actin 2. Relative transcript abundance of ATHB2, HAT1, HAT2, HAT3 and ATHB4 at each time point is normalized to their relative levels in DMSO-treated seedlings at the same time points. The standard deviation for all values is ≤0.2 (TIFF 1731 kb)

Supplementary Fig. 7

Root digital in situ analyses of the HD-Zip II genes. Expression data from Birnbaum et al. (2003) were deduced from and visualized by the Arabidopsis eFP Browser (Winter et al. 2007; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) (TIFF 6819 kb)

Supplementary Fig. 8

Shoot digital in situ analyses of the HD-Zip II genes. Expression data from Schmid et al. (2005) were deduced from Arabidopsis eFP Browser (Winter et al. 2007; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) and used to plot histograms. (a) Chart shows absolute expression levels of HD-Zip II genes in rosette leaves from 17 day-old seedlings. Expression levels in leaf #2, #4, #6, #8, #10 and #12 were depicted with differently green-colored bars; (b) Chart shows absolute expression levels of HD-Zip II genes in the shoot apices from 7 day-old (vegetative), 14 day-old (transition) and 21 day-old (inflorescence) seedlings (TIFF 1418 kb)

Supplementary Fig. 9

Flower digital in situ analyses of the HD-Zip II genes. Expression data in flower stages 12 and 15 from Schmid et al. (2005) were deduced from and visualized by the Arabidopsis eFP Browser (Winter et al. 2007; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Sep, sepals; pet, petals; stam, stamens; carpels (TIFF 3071 kb)

Supplementary Fig. 10

HD-Zip II genes’ expression in 35S::HAT1, 35S::HAT2 and 35S::ATHB2 plants. Total RNAs were extracted and subjected to qRT-PCR using UPL probes and primers specific to the genes indicated in the left of the histograms (TIFF 1998 kb)

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Ciarbelli, A.R., Ciolfi, A., Salvucci, S. et al. The Arabidopsis Homeodomain-leucine Zipper II gene family: diversity and redundancy. Plant Mol Biol 68, 465–478 (2008). https://doi.org/10.1007/s11103-008-9383-8

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