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Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29

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Abstract

HIPP26 from Arabidopsis thaliana belongs to a novel class of plant proteins, characterized by a heavy metal associated domain and an additional isoprenylation motif. It is induced during cold, salt and drought stress. The nuclear localization of HIPP26, predicted by a NLS motif, could be confirmed in onion epidermal cells overexpressing GFP-HIPP26. Experiments with modified HIPP26 indicate that the isoprenylation plays a role in the spatial distribution in the nucleus. Using promoter-GUS constructs, a tissue specific expression pattern of HIPP26 could be shown, with high expression in the vascular tissue. By a yeast-two-hybrid approach a strong interaction of HIPP26 with the zinc finger homeodomain transcription factor ATHB29, which is known to play a role in dehydration stress response could be detected. This was confirmed by GST pull-down assays. When using a modified HIPP26 lacking the two central cysteines of the heavy metal associated domain, ATHB29 was not bound in the GST pull-down assay, indicating that this structure is necessary for the interaction. Further yeast-two-hybrid analyses testing interaction of different members of the HIPP family with related zinc finger transcription factors revealed a specific interaction of ATHB29 with several HIPP proteins. A functional relationship between HIPP26 and ATHB29 is also indicated by experiments with mutants of HIPP26 showing altered expression levels of such genes known to be regulated by ATHB29.

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Abbreviations

GUS:

Glucuronidase

HIPP:

Heavy metal associated isoprenylated plant protein

HMA:

Heavy metal associated domain

NLS:

Nuclear localization signal

RTQ-PCR:

Quantitative RealTime PCR

smRS-GFP:

Solubility modified red shifted-green fluorescent protein

Y2H:

Yeast two hybrid

ZF-HD:

Zinc finger-homeodomain box

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Acknowledgements

We thank Ingrid Bauerfeld and Claudia Schramm for excellent technical assistance and Claudia Humbeck for her help during the preparation of the manuscript.

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Authors and Affiliations

  1. Institute of Biology, Martin-Luther-University Halle-Wittenberg, Weinbergweg 10, 06120, Halle, Germany

    Olaf Barth, Sebastian Vogt, Ria Uhlemann, Wiebke Zschiesche & Klaus Humbeck

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  1. Olaf Barth
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  2. Sebastian Vogt
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  3. Ria Uhlemann
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  5. Klaus Humbeck
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Correspondence to Klaus Humbeck.

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Supplemental Fig. 1

Alignment of the deduced amino acid sequences of HIPPs of subcluster III (see Fig. 1a) based on the sequence of the barley protein HvFP1. In addition the sequence of the heavy metal associated (HMA) domain (pfam00403.6) is shown. Boxes indicate the NLS (nuclear localization signal), the HMA domain including the central M/L/IxCxxC motif and the C-terminal isoprenylation motif (CaaX) which is accompanied by the cCT sequence. The black background indicates amino acid residues that are identical, and grey shading indicates amino acids that are similar to the HvFP1 sequence. Alignment was performed using ClustalW in the Lasergene expert sequence analysis software (DNASTAR Inc., Madison, WI, USA) (JPG 1609 kb)

Supplemental Fig. 2

Northern analysis of transcript levels of HIPP26 in homozygous F2 plants caring either the AtHIPP26 wildtype or the Athipp26 knock out alleles before and after 24 h of a combined cold (6°C) and high light (600 µEm−2 s−1) treatment. Four leaves of three different plants were pooled before total RNA was extracted. The identity numbers of each individual plant are indicated. Two different exposure times are shown. Ethidium bromide stained rRNA illustrates an equal loading of 15 µg total RNA per sample (JPG 1309 kb)

Supplemental Table S1

All the oligo nucleotides used for cloning and quantitative RealTime PCR procedures as described in the chapter material and methods (XLS 26 kb

Supplemental Table S2

Complete web signal scan results using the plant cis-acting regulatory DNA elements (PLACE) database (Higo et al. 1999, http://www.dna.affrc.go.jp/PLACE/) and a 1055 bp DNA sequence upstream the coding region of HIPP26. (XLS 80 kb)

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Barth, O., Vogt, S., Uhlemann, R. et al. Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29. Plant Mol Biol 69, 213–226 (2009). https://doi.org/10.1007/s11103-008-9419-0

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