Plant Molecular Biology

, Volume 68, Issue 4–5, pp 465–478 | Cite as

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

  • Angela Raffaella Ciarbelli
  • Andrea Ciolfi
  • Samanta Salvucci
  • Valentino Ruzza
  • Marco Possenti
  • Monica Carabelli
  • Alberto Fruscalzo
  • Giovanna Sessa
  • Giorgio Morelli
  • Ida Ruberti


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.


Arabidopsis Gene expression HD-Zip II gene family Light quality changes Leaf development Phylogenesis Shade avoidance response 



Binding site 1


Binding site 2






Differential Interference Contrast


Dimethyl sulfoxide


Homeodomain-Leucine Zipper


Indole Acetic Acid


Murashige and Skoog medium


Open Reading Frame


quantitative Reverse Transcription-Polymerase Chain Reaction


Reverse Transcription-Polymerase Chain Reaction




Universal Probe Library


Untranslated Region



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.

Supplementary material

11103_2008_9383_MOESM1_ESM.tif (2.2 mb)
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)
11103_2008_9383_MOESM2_ESM.tif (521 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)
11103_2008_9383_MOESM3_ESM.tif (3.4 mb)
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)
11103_2008_9383_MOESM4_ESM.tif (1.7 mb)
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)
11103_2008_9383_MOESM5_ESM.tif (1.9 mb)
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)
11103_2008_9383_MOESM6_ESM.tif (1.7 mb)
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)
11103_2008_9383_MOESM7_ESM.tif (6.7 mb)
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; (TIFF 6819 kb)
11103_2008_9383_MOESM8_ESM.tif (1.4 mb)
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; 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)
11103_2008_9383_MOESM9_ESM.tif (3 mb)
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; Sep, sepals; pet, petals; stam, stamens; carpels (TIFF 3071 kb)
11103_2008_9383_MOESM10_ESM.tif (2 mb)
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)
11103_2008_9383_MOESM11_ESM.doc (38 kb)
(DOC 37 kb)
11103_2008_9383_MOESM12_ESM.doc (45 kb)
(DOC 45 kb)
11103_2008_9383_MOESM13_ESM.doc (38 kb)
(DOC 38 kb)


  1. Aoyama T, Dong CH, Wu Y, Carabelli M, Sessa G, Ruberti I et al (1995) Ectopic expression of the Arabidopsis transcriptional activator Athb-1 alters leaf cell fate in tobacco. Plant Cell 7:1773–1785PubMedCrossRefGoogle Scholar
  2. Baima S, Possenti M, Matteucci A, Wisman E, Altamura M, Ruberti I et al (2001) The Arabidopsis ATHB8 HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristems. Plant Physiol 126:643–6555. doi: 10.1104/pp.126.2.643 PubMedCrossRefGoogle Scholar
  3. Bechtold N, Ellis JE, Pellettier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris 316:1194–1199Google Scholar
  4. Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW et al (2003) A gene expression map of the Arabidopsis root. Science 302:1956–1960. doi: 10.1126/science.1090022 PubMedCrossRefGoogle Scholar
  5. Blanc G, Hokamp K, Wolfe KH (2003) A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis genome. Genome Res 13:137–144. doi: 10.1101/gr.751803 PubMedCrossRefGoogle Scholar
  6. Bowman JL (2004) Class III HD-Zip gene regulation, the golden fleece of ARGONAUTE activity? Bioessays 26:934–938. doi: 10.1002/bies.20103 CrossRefGoogle Scholar
  7. Byrne ME (2006) Shoot meristem function and leaf polarity: the role of class III HD-ZIP genes. PLoS Genet 2:e89. doi: 10.1371/journal.pgen.0020089 PubMedCrossRefGoogle Scholar
  8. Carabelli M, Sessa G, Baima S, Morelli G, Ruberti I (1993) The Arabidopsis Athb-2 and -4 genes are strongly induced by far-red-rich light. Plant J 4:469–479. doi: 10.1046/j.1365-313X.1993.04030469.x PubMedCrossRefGoogle Scholar
  9. Carabelli M, Morelli G, Whitelam G, Ruberti I (1996) Twilight-zone and canopy shade induction of the Athb-2 homeobox gene in green plants. Proc Natl Acad Sci USA 93:3530–3535. doi: 10.1073/pnas.93.8.3530 PubMedCrossRefGoogle Scholar
  10. Carabelli M, Possenti M, Sessa G, Ciolfi A, Sassi M, Morelli G et al (2007) Canopy shade causes a rapid and transient arrest in leaf development through auxin-induced cytokinin oxidase activity. Genes Dev 21:1863–1868. doi: 10.1101/gad.432607 PubMedCrossRefGoogle Scholar
  11. Devlin PF, Yanovsky MJ, Kay SA (2003) A genomic analysis of the shade avoidance response in Arabidopsis. Plant Physiol 133:1617–1629. doi: 10.1104/pp.103.034397 PubMedCrossRefGoogle Scholar
  12. Felsenstein J (1989) PHYLIP: phylogeny inference package version 3.2. Cladistics 5:164–166Google Scholar
  13. Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, SeattleGoogle Scholar
  14. Franklin KA, Praekelt U, Stoddart WM, Billingham OE, Halliday KJ, Whitelam GC (2003) Phytochromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiol 131:1340–1346. doi: 10.1104/pp.102.015487 PubMedCrossRefGoogle Scholar
  15. Green PJ (1993) Control of mRNA stability in higher plants. Plant Physiol 102:1065–1070PubMedGoogle Scholar
  16. Henriksson E, Olsson AS, Johannesson H, Johansson H, Hanson J, Engstrom P et al (2005) Homeodomain leucine zipper class I genes in Arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiol 139:509–518. doi: 10.1104/pp.105.063461 PubMedCrossRefGoogle Scholar
  17. Horiguchi G, Kim GT, Tsukaya H (2005) The transcription factor AtGRF5 and the transcription co-activator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana. Plant J 43:68–78. doi: 10.1111/j.1365-313X.2005.02429.x PubMedCrossRefGoogle Scholar
  18. Meijer AH, de Kam RJ, d’Erfurth I, Shen W, Hoge JH (2000) HD-Zip proteins of families I and II from rice: interactions and functional properties. Mol Gen Genet 263:12–21. doi: 10.1007/PL00008671 PubMedCrossRefGoogle Scholar
  19. Morelli G, Ruberti I (2000) Shade avoidance responses. Driving auxin along lateral routes. Plant Physiol 122:621–626. doi: 10.1104/pp.122.3.621 PubMedCrossRefGoogle Scholar
  20. Morelli G, Ruberti I (2002) Light and shade in the photocontrol of Arabidopsis growth. Trends Plant Sci 7:399–404. doi: 10.1016/S1360-1385(02)02314-2 PubMedCrossRefGoogle Scholar
  21. Morelli G, Baima S, Carabelli M, Di Cristina M, Lucchetti S, Sessa G et al (1998) Homeodomain-leucine zipper proteins in the control of plant growth and development. In: Last R, Lo Schiavo F, Morelli G, Raikel N (eds) Cellular integration of signaling pathways in plant development, vol H 104, pp 251–262. Springer Verlag, NATO-ASI seriesGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue coltures. Physiol Plant 15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  23. Nakamura M, Katsumata H, Abe M, Yabe N, Komeda Y, Yamamoto KT et al (2006) Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis. Plant Physiol 141:1363–1375. doi: 10.1104/pp.106.077388 PubMedCrossRefGoogle Scholar
  24. Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol 2:e258. doi: 10.1371/journal.pbio.0020258 PubMedCrossRefGoogle Scholar
  25. Ohgishi M, Oka A, Morelli G, Ruberti I, Aoyama T (2001) Negative autoregulation of the Arabidopsis homeobox gene ATHB-2. Plant J 25:389–398. doi: 10.1046/j.1365-313x.2001.00966.x PubMedCrossRefGoogle Scholar
  26. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007. doi: 10.1093/nar/29.9.e45 CrossRefGoogle Scholar
  27. Picard D, Salser SJ, Yamamoto KR (1988) A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell 54:1073–1080. doi: 10.1016/0092-8674(88)90122-5 PubMedCrossRefGoogle Scholar
  28. Ruberti I, Sessa G, Lucchetti S, Morelli G (1991) A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. EMBO J 10:1787–1791PubMedGoogle Scholar
  29. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic tree. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  30. Sawa S, Ohgishi M, Goda H, Higuchi K, Shimada Y, Yoshida S et al (2002) The HAT2 gene, a member of the HD-Zip gene family, isolated as an auxin inducible gene by DNA microarray screening, affects auxin response in Arabidopsis. Plant J 32:1011–1022. doi: 10.1046/j.1365-313X.2002.01488.x PubMedCrossRefGoogle Scholar
  31. Schena M, Lloyd AM, Davis RW (1993) The HAT4 gene of Arabidopsis encodes a developmental regulator. Genes Dev 7:367–379PubMedCrossRefGoogle Scholar
  32. Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M et al (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37:501–506. doi: 10.1038/ng1543 PubMedCrossRefGoogle Scholar
  33. Sessa G, Morelli G, Ruberti I (1993) The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO J 12:3507–3517PubMedGoogle Scholar
  34. Sessa G, Carabelli M, Ruberti I, Baima S, Lucchetti S, Morelli G (1994) Identification of distinct families of HD-Zip proteins in Arabidopsis thaliana. In: Puigdomenech P, Coruzzi G (eds) Molecular-genetic analysis of plant development and metabolism. NATO-ASI series, vol H 81, pp 411–426. Springer VerlagGoogle Scholar
  35. Sessa G, Morelli G, Ruberti I (1997) DNA-binding specificity of the homeodomain-leucine zipper domain. J Mol Biol 274:303–309. doi: 10.1006/jmbi.1997.1408 PubMedCrossRefGoogle Scholar
  36. Sessa G, Carabelli M, Sassi M, Ciolfi A, Possenti M, Mittempergher F et al (2005) A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev 19:2811–2815. doi: 10.1101/gad.364005 PubMedCrossRefGoogle Scholar
  37. Steindler C, Borello U, Carabelli M, Morelli G, Ruberti I (1997) Phytochome A, phytochome B and other phytochome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants. Plant Cell Environ 20:759–763. doi: 10.1046/j.1365-3040.1997.d01-123.x CrossRefGoogle Scholar
  38. Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T et al (1999) Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression. Development 126:4235–4245PubMedGoogle Scholar
  39. Sullivan ML, Green PJ (1993) Post-transcriptional regulation of nuclear-encoded genes in higher plants: the roles of mRNA stability and translation. Plant Mol Biol 23:1091–1104. doi: 10.1007/BF00042344 PubMedCrossRefGoogle Scholar
  40. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi: 10.1093/nar/22.22.4673 PubMedCrossRefGoogle Scholar
  41. Tiwari SB, Hagen G, Guilfoyle TJ (2004) Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell 16:533–543. doi: 10.1105/tpc.017384 PubMedCrossRefGoogle Scholar
  42. Triezenberg SJ, Kingsbury RC, McKnight SL (1988) Functional dissection of VP16, the transactivator of herpes simplex virus immediate early gene expression. Genes Dev 2:718–729. doi: 10.1101/gad.2.6.718 PubMedCrossRefGoogle Scholar
  43. Wagner A (2001) Birth and death of duplicated genes in completely sequenced eukaryotes. Trends Genet 17:237–239. doi: 10.1016/S0168-9525(01)02243-0 PubMedCrossRefGoogle Scholar
  44. Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  45. Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An “electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS One 2:e718. doi: 10.1371/journal.pone.0000718 PubMedCrossRefGoogle Scholar
  46. Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632. doi: 10.1104/pp.104.046367 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Angela Raffaella Ciarbelli
    • 1
  • Andrea Ciolfi
    • 1
  • Samanta Salvucci
    • 1
  • Valentino Ruzza
    • 1
  • Marco Possenti
    • 2
  • Monica Carabelli
    • 1
  • Alberto Fruscalzo
    • 1
  • Giovanna Sessa
    • 1
  • Giorgio Morelli
    • 2
  • Ida Ruberti
    • 1
  1. 1.Institute of Molecular Biology and PathologyNational Research CouncilRomeItaly
  2. 2.National Research Institute for Food and NutritionRomeItaly

Personalised recommendations