Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta) Vanessa R. Falcão & Mariana C. Oliveira & Pio Colepicolo Received: 14 October 2009 / Revised and accepted: 5 January 2010 / Published online: 28 January 2010 # Springer Science+Business Media B.V. 2010 Abstract The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economical- ly important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biolog- ical clock at the mRNA levels for NR in G. tenuistipitata. Keywords Biological rhythm . Phylogeny . Gene expression . Gracilaria tenuistipitata . Nitrate reductase Introduction The major source of nitrogen in the marine ecosystem is in the form of nitrate (Adams and Mortenson 1985), which must be reduced to ammonia or amine to be assimilated into the biosynthesis of nitrogen-containing compounds such as proteins and nucleic acids (Crawford and Campbell 1990; Solomonson and Barber 1990). The reduction of nitrate to nitrite is the first step in the reduction to ammonia; it is catalyzed by a NAD(P)H- dependent enzyme named nitrate reductase (NR; EC 1.6.6.1), and it is considered to be rate-limiting in the nitrate assimilation process (Lopes et al. 1997; Ramalho et al. 1995). Nitrate reductase is a high-molecular-weight complex protein with three prosthetic groups, FAD, heme (cytochrome b 557 ), and Mo-pterin, which uses NADH or NADPH as the electron donor (Caboche and Rouzé 1990; Crawford 1995), and it is subject to tight controls at the levels of enzyme activity, synthesis, and degradation (Lillo et al. 2001). NR protein has been purified from several organisms, and it occurs in a wide variety of molecular weights as well as number of subunits. NR size ranges from 200 kDa in spinach, with two subunits (Hewitt 1975), to 500 kDa in the green alga Ankistrodesmus braunii, with eight subunits of 58 kDa (De la Rosa et al. 1981). In response to diurnal changes in photosynthesis, NR expression and activity vary between day and night (Stitt et al. 2002). Analysis of NR genes in plants showed that their expressions were primarily regulated at the transcriptional level (Campbell 1999). The expression of NR is a complex process regulated at the transcriptional level by different factors such as nitrate, CO 2 , light, and V. R. Falcão : P. Colepicolo Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo( CP 20780, CEP 05599-970, Brazil M. C. Oliveira Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, São Paulo CEP 05508-900, Brazil V. R. Falcão (*) Department of Plant Cellular and Molecular Biology, Ohio State University, 318W 12th Avenue, Aronoff Laboratory, Room 500, Columbus, OH 43210, USA e-mail: vanessa.falcao@gmail.com J Appl Phycol (2010) 22:613–622 DOI 10.1007/s10811-010-9501-2