Oxidative Stress In Fugacium Kawagutii Induce By Elevated Temperature And Nutrient Deficiency

Coral reefs provide habitats for many marine species.Although located in oligotrophic waters,coral reefs show high primary productivity.The organic carbon fixed by coral reefs not only support massive coral lives but also commercial and recreational fisheries.Corals rely on their symbiotic photosynthetic dinoflagellates from the family of Symbiodiniaceae for photosynthetic products while Symbiodiniaceae species rely on habitats and nutrients provided by corals.Many studies have shown that the coral-Symbiodiniaceae symbiosis is vulnerable to environmental changes including global warming,nutrient limitation,and the varying salinity and light conditions.Thermal stress and other environmental insults are able to trigger photoinhibition,oxidative damage,and the breakage of symbiosis,leading to coral bleaching.The adverse process is likely caused by the excessive reactive oxidase species(ROS)generated by the symbionts,which will have detrimental effect on host cells.Excess production of ROS by the endosymbionts is commonly blamed for coral bleaching as a result of thermal stress and other environmental insults.The quantity of ROS produced and its gene expression level often differ under different stress,suggesting that different mechanisms are at play.Nevertheless,effects of environmental factors including the varying temperatures and nutrients on ROS dynamics are not so well documented and the molecular mechanism remains elusive.Here,we used Fugacium kawagutii as a model organism and investigated its growth,photosynthetic efficiency,cell size,dissolved inorganic phosphorus(DIP)concentration,ROS production,and NADPH oxidase(NOX)gene expression when the alga was exposed to high temperature,phosphorus(P)limitation,and nitrogen(N)limitation.To enable proper quantification of gene expression,identification of reference genes is essential.These kind of genes would show stable expression levels under varying growth conditions.The commonly used reference genes such as 18S ribosomal RNA(18S rRNA)and glyceraldehyde-3-phosphate dehydrogenase(gapdh)are not always stable under different treatment conditions and in different cell types.In this study,we obtained the most stable housekeeping gene under different conditions in F.kawagutii through screening 11 potential reference genes including Glyceraldehyde-3phosphate dehydrogenase(GAPDH),S-adenosyl-L-methionine synthetase(SAM),Cytochrome oxidase subunit 1(Cox),Calmodulin(Cal),Ribosomal protein S4(Rp-s4),Actin(Act),Cyclophin(Cyc),Beta-Tublin(Tub),Heat shock protein 90(Hsp),Ubiquitin(Ubiq)and proliferating cell nuclear antigen(PCNA).The major experiments and results are as follows:1.Other conditions being equal,for the temperature experiment,32? was set as a treatment group(high temperature),and 25? was set as a control group,for quadruple cultures at each temperature.Cell concentration,photosynthesis efficiency,and ROS production were measured throughout the whole experimental period.In F.kawagutii(Symbiotic Symbiodiniaceae),we observed that the cell concentration of the treatment group was significantly lower than the control group after 96 hours,while the overall photosystem ? quantum efficiency(Fv/Fm)increased in the treatment group.Furthermore,intracellular accumulation of ROS was observed in the the treatment group after being exposed to 32? for 2 hours and then recovered for 24 hours.However,NOX expression was surprisingly inhibited after heat shock,especially after 24 hours,indicating that the observed ROS accumulation was not due to elevation of NADPH oxidase-dependent ROS production.The quick recovery to lower ROS might be due to up-regulation of anti-oxidative capacity conferring thermal tolerance to F.kawagutii.In Effrenium voratum(Free-living Symbiodiniaceae),cells of treatment group could maintain growth after 24 hours,and then the growth slowed down;ROS was always significantly lower than the control group.In addition,we found that ROS production increased from the end of the dark period to the beginning of the light period indicating a stimulatory role of dark to light switch for ROS production.2.In the study of the effects of N limitation on F.kawagutii intracellular ROS level and NOX expression level,we measured the cell concentration,Fv/Fm,cell size,intracellular ROS level,and NOX gene expression.Results showed that under Ndeprived condition,F.kawagutii growth decreased,cells size significantly enlarged,while photosystem ? quantum efficiency decreased but not significantly.Under this condition,intracellular ROS levels increased significantly while gene expression levels of NOX increased but not significantly.The results suggest that consistent with previous studies,under N deficiency cells could continue to photosynthesize and grow but not divide,resulting in cell enlargement,but antioxidant capacity decreased,leading to accumulation of ROS in the cells.3.In exploring the effect of P deficiency on F.kawagutii intracellular ROS level and NOX expression level,we measured the cell concentration,Fv/Fm,DIP concentration in the medium,intracellular ROS level,and NOX gene expression.Results showed that the DIP concentration in the P-deprived group closed to 0,and population growth decreased significantly.Both ROS levels and gene expression levels of NOX were down-regulated albeit not significantly.These results suggest that P deficiency has little effect on the production of ROS in F.kawagutii.We speculate that this could be because F.kawagutii has developed an adaptive strategy for P deficiency and is able to control ROS production at a lower level.4.Eleven genes commonly used as reference genes in various organism were selected for reference gene screening in F.kawagutii under different culture conditions.GeNorm and NormFinder were used for evaluating the expression stabilities of these candidate reference genes.GAPDH and PCNA were found to exhibit stable expression levels across different temperature and N as well as P nutrient conditions.This result will be helpful for future studies on the effect of different growth conditions on gene expression in F.kawagutii and possibly other dinoflagellate species as well.