Corals on a nitrate diet: feeding or failing? Unlocking the secrets of nitrate assimilation by Symbiodiniaceae from reef-building corals and its role in holobiont response to thermal stress

Coral reefs have fascinated both the public and scientists for decades due to their ability to thrive in nutrient-poor waters while sustaining high primary productivity and rich biodiversity. Their evolutionary success is largely attributed to the unique biology of reef-building corals, which live in close symbiosis with dinoflagellates from the family Symbiodiniaceae. These photosynthetic endosymbionts assimilate inorganic carbon and nitrogen into organic molecules such as carbohydrates, amino acids, and lipids, which are then translocated to the cnidarian host. This relationship enables corals to meet their energy needs for tissue and skeletal growth in oligotrophic tropical environments. However, increasing environmental and anthropogenic pressures fragilize this delicate partnership, with the well-documented phenomenon of coral bleaching, where corals lose their photosynthetic symbionts. Thermal stress due to climate change is recognized as one of the most significant threats to coral reefs. Yet, the combined effects of other environmental factors remain poorly understood. Enrichment of dissolved inorganic nitrogen (DIN), particularly nitrate, is suspected to exacerbate coral stress responses and compromise coral survival. However, evidence on this issue is complex and sometimes equivocal across studies, highlighting the need for further research into DIN assimilation by coral and its impact on holobiont stress responses. This research aimed to address these knowledge gaps by investigating nitrate assimilation by Symbiodiniaceae and examining the effects of nitrate enrichment on coral physiology, microbiome, and stress responses through a comprehensive approach that integrates molecular, physiological and microbial perspectives.
Coral hosts lack the necessary enzymes to reduce nitrate (NO3-) to ammonium (NH4+) for incorporation into biological molecules, whereas the photosynthetic Symbiodiniaceae express nitrate reductase (NR). However, evidence for active nitrate reduction by the algae during symbiosis is limited and primarily based on nitrate uptake measurements, which may not accurately reflect nitrate assimilation by the symbionts. Using western blotting and qRT-PCR, we studied NR expression in cultured Symbiodiniaceae under various conditions to understand its regulation mechanisms. We found that NR protein expression is dynamic and reversible, influenced by NO3- and NH4+ concentrations. Specifically, NR protein synthesis is induced by NO3- and downregulated in the presence of NH4+. Additionally, NR protein synthesis depends on light exposure and the integrity of the photosynthetic electron transport chain. qRT-PCR assays suggested that NR expression is largely regulated post-transcriptionally, as the NR-coding gene is uniformly expressed across conditions. We further examined NR protein expression in in hospite Symbiodiniaceae of three coral species, which were nitrogen-depleted before exposure to a NO3- enrichment. We demonstrated that Symbiodiniaceae express NR during symbiosis, and that this expression is dependent on NO3- concentrations.
While nitrate sustains symbiont communities, it has also been reported to adversely affect responses to oxidative stress and exacerbate bleaching. Using a crossed treatment experimental design in a mesocosm setup, we investigated the individual and combined effects of heat stress and nitrate enrichment on the physiological performance and the microbiome of the Great Barrier Reef coral Acropora kenti. Over four weeks, coral nubbins were exposed 5 µM NO3- enrichment and 5 DHW temperature stress. Elevated temperatures induced moderate to severe bleaching, unaffected by NO3- enrichment, with a concurrent shift in Cladocopium symbiont community composition. The loss of symbionts in heat-stressed corals was reflected in significantly diminished primary productivity (photosynthesis rates), respiration, and coral growth rates compared to controls, but these changes were not significantly different from the intermediary responses observed in nitrate-enriched corals at ambient temperatures. Moderate photoinhibition of heat-stressed corals occurred in the final days of the experiment, with no significant increase in non-photochemical quenching, suggesting oxidative stress in the symbionts was not the primary cause of bleaching. A. kenti’s microbiome was influenced by both host genotype and treatments, particularly heat stress, leading to higher dispersion in microbial community composition, suggesting a shift to a dysbiotic state. Nitrate enrichment induced significant differences in coral microbiome communities only under ambient temperatures, not heat stress. This study demonstrates that thermal stress is the primary driver of physiological and microbial changes in Acropora kenti, while nitrate enrichment did not affect coral thermal tolerance and had limited impacts on holobiont metabolism, symbiont photosynthetic efficiency, and the microbiome.
Overall, this research enhances our understanding of nitrate assimilation by coral symbionts and provides nuanced insights into the effects of nitrate on coral holobiont functioning.