Wang, Yanfei2017-02-032017-02-032016http://udspace.udel.edu/handle/19716/20387Chattonella subsalsa is a harmful alga that can form fish-killing blooms and cause severe damage to the ecosystem. In Delaware Inland Bays, C. subsalsa has formed mixed blooms with other species in recent years. The reason for the persistence of these blooms and the capacity for these species to avoid competitive exclusion remains unknown. Nitrogen is a limiting source in the aquatic environment, and its input may stimulate blooms dominated by C. subsalsa. Therefore, competing for nitrogen source may contribute to the success and survival of this species. For organisms to use nitrate as a nitrogen source, nitrate reductase catalyzes the first and also rate limiting step in nitrate assimilation. Algal nitrate reductase is responsive to nitrogen source, temperature, light intensity and its endogenous diel rhythm. In plants, it is also regulated by reversible phosphorylation of a conserved serine residue in the hinge 1 region, and sequential binding of 14-3-3 proteins at the post-translational level. However, 14-3-3 binding motifs within nitrate reductase were only found in plants, but not in algae. Previous research found a novel nitrate reductase, NR2-2/2HbN (NR2), in C. subsalsa, and this enzyme has a 2/2 hemoglobin domain within its hinge 2 region. In this research, another novel nitrate reductase, NR3, was found in this alga, and its sequence indicates the presence of a 14-3-3 binding motif in the hinge 1 region. To date, this is the first report for the presence of the 14-3-3 binding motif in algal nitrate reductase. In Chapter 2, the sequence of NR3 was analyzed and compared with nitrate reductase sequences in algae and plants. The presence of a putative 14-3-3 binding motif in this enzyme was discussed. The expression and activity of nitrate reductase in C. subsalsa were measured in response to light, nitrogen source, and temperature. The results indicate that, at the gene expression level, both NR2 and NR3 were regulated by light and nitrogen source, while only NR2 was regulated by temperature. At the protein translational level, evidence was provided that NR activity was regulated by nitrogen and temperature by reversible phosphorylation and binding of 14-3-3 proteins, while NR activity in response to light may be regulated by alternative mechanisms. In Chapter 3, natural C. subsalsa blooms were stimulated by different nitrogen sources in two mesocosm experiments. One pulse of nitrogen was added to the first mesocosm experiment, while repeated pulses of nitrogen along with phosphate were added to the second mesocosm experiment. The growth rate of C. subsalsa and the entire assemblage, as well as NR2 and NR3 expression, were tested in order to investigate the implications of NR expression to the competition and survival of C. subsalsa in a dynamic environment. The results indicate that C. subsalsa out-competed other species with a low nitrogen concentration. Several strategies for the survival and success of C. subsalsa in the low nitrogen-loading environment were proposed based on the results: 1). C. subsalsa performed surge uptake and could store nitrogen; 2). C. subsalsa was capable of utilizing nitrate produced by nitrogen fixers or released by dead cells; 3). C. subsalsa regulates NR2 and NR3 expression differentially in response to different nitrogen conditions, such that NR2 may benefit C. subsalsa in favorable environments with a high concentration of nitrate, while NR3 may benefit C. subsalsa in a more dynamic and unfavorable environment with ammonium as the dominant nitrogen source.Nitrate reductases -- Delaware.Chattonella subsalsa -- Delaware.Raphidophyceae -- Delaware.Marine algae -- Delaware.Algal blooms -- Delaware.Gene expression.Thesis971244701