Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/21/2004
Publication Date: 8/1/2004
Citation: Ross, E.H., Elowsky, C., Stone, J., Arredondo, P.R., Sarath, G. 2004. Rice nonsymbiotic hemoglobin-2promoter is activated by the cytokinin-regulated transcription factor, arr-1. J Exp Bot. 55:1721-1731. Interpretive Summary: Nonsymbiotic hemoglobins are plant proteins now known to be present in all plant phyla . Although much has been learned about their biochemical properties using recombinant technologies, functional properties of these enigmatic proteins in plant cells have been slow to emerge. Studies in our laboratory have shown that in rice, nonsymbiotic hemoglobins are synthesized in cells and tissues undergoing some form of programmed cell death. These results and those from other researchers indicated a potential regulation of nonsymbiotic hemoglobin gene expression by plant hormones. In order to more fully understand these interactions, we computer-analyzed the promoter sequences of available rice hemoglobin genes and discovered conserved sequences that indicated responsiveness to hormones, most notably to cytokinins. We cloned the promoter region for one of the rice hemoglobin genes, and used this construct to understand its responsiveness to cytokinins using several model systems. All of our data shown in this manuscript document a direct interaction of the rice hemoglobin-promoter to cytokinin and cytokinin response regulatory proteins, specifically ARR1. This is the first report that shows the pathway involved in regulating gene expression of a nonsymbiotic hemoglobin.
Technical Abstract: Using in silico methods, we report the identification of several putative cis-elements in the rice (Oryza sativa) nonsymbiotic hemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters that confer phytohormone responsiveness. The rice NSHB2 promoter drove GUS expression in a tissue-specific pattern in Arabidopsis thaliana. GUS expression included roots, vasculature of young leaves, flowers, and the abscission zone of the pedicel. In transient assays, activation of the rice NSHB2 promoter was significantly upregulated in the presence of the cytokinin 6-benzylaminopurine (BA). Deletion analysis indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-activated ARR1-binding element significantly decreased promoter activity in the presence of cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type NSHB2 promoter activity, but not the activity of the mutated promoter in the absence of cytokinin. Expression of ARR1 in the presence of cytokinin resulted in the highest level of activation of the wild-type promoter. Moderate activation of the mutated promoter was observed only in the presence of cytokinin and ARR1, indicating that the rice NSHB2 promoter is regulated by the ARR1 protein, and potentially 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.