COMPARISON OF NITRATE REDUCTASE SEQUENCE AND EXPRESSION PATTERNS FOR THE
HARMFUL RAPHIDOPHYTES, Heterosigma akashiwo AND Chattonella subsalsa
Kathryn J. Coyne1§, Jennifer J. Stewart1 and Mark A. Warner1
1
University of Delaware College of Earth, Ocean, and Environment, 700 Pilottown Rd., Lewes, DE 19958
kcoyne@udel.edu
§
The raphidophytes Heterosigma akashiwo and Chattonella subsalsa bloom to high density annually in the
Delaware Inland Bays (DIB), DE. As part of an effort to understand the impacts of environmental factors on
bloom dynamics, we evaluated differences in nitrate utilization for these two species. Previous research
demonstrated that the DIB isolate of H. akashiwo was able to maintain maximum growth at nitrate levels that
were an order of magnitude lower than required for C. subsalsa. Other research, however, showed that H.
akashiwo was a poor competitor over a range of nitrogen concentrations when combined with other HAB
species in static laboratory culture. We hypothesized that H. akashiwo may be a better competitor under
dynamic conditions and is better able to tolerate and respond to changes in the environment when compared to
other HAB species. To understand species-specific capabilities to assimilate nitrogen under varying
environmental conditions, we evaluated nitrate reductase (NR) gene expression in DIB isolates of H. akashiwo
and C. subsalsa in laboratory culture manipulations. Nitrate reductase catalyzes the rate-limiting step in nitrate
assimilation and is regulated at the level of gene transcription. NR gene expression patterns in vascular plants,
green algae and marine diatoms, however, are highly variable depending on nitrogen status of the cell. In
addition, little is known about induction and regulation of NR expression in other phytoplankton species. Here,
we evaluated and compared NR sequence and expression patterns in laboratory cultures of H. akashiwo and C.
subsalsa in relation to changes in temperature, light intensity and nitrogen source. Phylogenetic analysis of NR
sequences from raphidophytes suggests that they are distinct from other plant and algal nitrate reductases. For
laboratory culture experiments, nucleic acids were extracted and NR transcript abundance for Heterosigma and
Chattonella were evaluated using quantitative reverse transcriptase real-time PCR. Our research demonstrates
species-specific differences in regulation of NR expression. Results of this research will be discussed with
respect to the competitive dynamics and bloom formation for these two HAB species.