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JXB Advance Access originally published online on July 16, 2004
Journal of Experimental Botany 2004 55(403):1721-1731; doi:10.1093/jxb/erh211
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Journal of Experimental Botany, Vol. 55, No. 403, © Society for Experimental Biology 2004; all rights reserved

RESEARCH PAPER

Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1

Emily J. H. Ross1,*, Julie M. Stone1, Christian G. Elowsky2, Raul Arredondo-Peter3, Robert V. Klucas1 {dagger} and Gautam Sarath4

1Department of Biochemistry and Plant Science Initiative, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, USA
2Johns Hopkins University, School of Medicine, Division of Gastroenterology, 720 Rutland Ave., Baltimore, MD 21205, USA
3Laboratorio de Biofisica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, 62210 Cuernavaca, Morelos, México
4USDA-ARS, Wheat, Sorghum and Forage Unit, University of Nebraska-Lincoln, Lincoln, Nebraska, USA

* Present address and to whom correspondence should be sent: Department of Entomology, Plant Industry 306, University of Nebraska-Lincoln, Lincoln, NE 68583, USA. Fax: +1 402 472 4687. E-mail: eross2{at}unl.edu

Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.

Key words: ARR proteins, cytokinins, gene regulation, non-symbiotic plant haemoglobin


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