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Phosphorylation of Arabidopsis response regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act as a negative regulator of cytokinin signaling

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Abstract

Cytokinins are plant hormones that regulate diverse aspects of plant growth and development. Arabidopsis cytokinin signal transduction utilizes a multi-step two-component signaling (TCS) system by histidyl–aspartidyl phosphorelays. We here show that phosphorylation of ARR7, an A-type response regulator that acts as a negative regulator of cytokinin signaling, is required for its function in plants. Phosphorylation of ARR7 is inhibited in vitro by mutation in a putative phospho-accepting Asp residue into an Asn residue (ARR7D85N). While ectopic expression of ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of ARR7 D85N at the similar level does not generate these phenotypes. ARR7D85N is localized to the nucleus and the half-life of this mutant protein is similar to that of ARR7 in Arabidopsis mesophyll protoplasts. These results suggest that the phosphorylation of ARR7 is necessary for ARR7-mediated cytokinin response.

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Abbreviations

AHK:

Arabidopsis histidine kinase

AHP:

Arabidopsis histidine-containing phosphotransfer domain protein

ARR:

Arabidopsis response regulator

BA:

6-Benzyladenine

CKI:

Cytokinin insensitive

CRE:

Cytokinin response

ETR:

Ethylene resistant

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

HPt:

Histidine-containing phosphotransfer protein

LUC:

Luciferase

MS:

Murashige Skoog

MUG:

4-Methylumberlliferyl-β-d-glucuronide

NLS:

Nuclear localization signal

PEG:

Polyethylene glycol

TCS:

Two-component signaling

WOL:

Wooden leg

2,4-D:

2,4-Dichlorophenoxyacetic acid

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Acknowledgments

We thank Enamul Huq (University of Texas at Austin, USA) for pTLNLS vector and Illdoo Hwang (POSTECH, Pohang, Korea) for pDJ301 vector. We also thank Jin-Young Park for preliminary experiments. This work was supported by grants from the Agricultural Plant Stress Research Center (R11-2001-092-04001-0) funded by the Korea Science and Engineering Foundation and from the Plant Diversity Research Center of 21st Century Frontier Research Program (PF06302-01) funded by the Ministry of Science and Technology of the Korean Government to J. Kim.

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  1. Department of Plant Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Buk-Gu, Gwangju, 500-757, South Korea

    Dong Ju Lee, Sunmi Kim, Young-Min Ha & Jungmook Kim

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  1. Dong Ju Lee
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  2. Sunmi Kim
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Correspondence to Jungmook Kim.

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Lee, D.J., Kim, S., Ha, YM. et al. Phosphorylation of Arabidopsis response regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act as a negative regulator of cytokinin signaling. Planta 227, 577–587 (2008). https://doi.org/10.1007/s00425-007-0640-x

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