Deciphering the expression and regulation of nitrate reductase by Symbiodiniaceae in culture and in hospite

Coral holobionts are highly efficient in the assimilation of nitrogen through heterotrophic feeding or the uptake of dissolved inorganic nitrogen. Although NO3- is the most abundant nitrogen source in the ocean, corals preferably uptake NH4+ due to its reduced state and energetically favorable assimilation. However, in conditions of low availability of environmental NH4+, coral holobionts are capable of depleting environmental NO3-. Symbiodiniaceae are vital partners of the symbiosis for nutrient assimilation. In addition to providing translocated photosynthates, they facilitate the acquisition of environmental nitrogen and account for most of the uptake of dissolved inorganic nitrogen. While the uptake of NH4+ by the coral host and its symbiotic partners is a well-known process, NO3- assimilation remains poorly studied. Coral hosts are incapable of nitrate reduction as they do not possess the necessary enzymes, while Symbiodiniaceae have been shown to express the enzyme nitrate reductase (NR). However, the evidence supporting the active reduction of nitrate by the symbiotic algae during symbiosis is scarce and equivocal. Rigorous studies on nitrate assimilation by Symbiodiniaceae are lacking yet essential for the understanding of coral holobiont functioning. We studied the expression and regulation of NR in monoclonal and axenic Symbiodiniaceae cultures (SSB01 Breviolum minutum and Symbiodinium microadriaticum) on different N sources (NO3-, NH4+, NO3- + NH4+). The algae expressed NR in culture, but the enzyme expression was repressed in the presence of NH4+ even when NO3- was available. Protein expression kinetics as well as NH4+ concentration threshold for NO3- assimilation inhibition were investigated using increasing NH4+ concentrations on NO3- medium. Following the addition of NH4+ to the medium, NR was actively degraded under 6 hours. In the absence of NH4+ and following the addition of NO3-, NR was synthesized over 3 to 6 hours. This illustrates the inhibition effect of NH4+ on NR expression. Symbiodiniaceae in culture are capable of utilizing environmental NO3-. However, the symbiotic algae could see their activity hindered by the host cellular environment and cellular NH4+ concentrations. These results raise questions about the occurrence of the enzyme NR in symbiosis along with the process of NO3- assimilation by coral holobionts.