Studies On Carbon Assimilation Mechanism Of A Typical Algal Bloom Species Prorocentrum Donghaiense

Prorocentrum donghaiense is a representative phytoplankton species of harmful algal blooms in the coastal waters of China.This species frequently causes a large-scale algal bloom in the southeast coast of China,which not only causes serious damage to marine ecosystem but also causes serious harm to marine organisms,aquaculture and public health.Although much effort has been devoted to its morphology,distribution,physiological-ecology,molecular identification and expression sequences,no study on the mechanism of carbon assimilation has been conduected,which impedes our understanding of the formation mechanism of its bloom to a certain extent.This thesis selected P.donghaiense as the model species and studied carbon assimilation mechanism and its response to environmental changes using a combination of real-time quantitative PCR,stable isotope mass ratio spectrometry,and physiological-ecological techniques.Expressions of key genes involved in C3,C4 pathways under different CO2 concentrations,temperatures and nitrogen condition were compared.Combined with the analysis of assimilation enzyme activities and stable isotope abundances of particulate organic carbon,we discussed the reponse mechanisms of carbon assimilation of P.donghaiense to environment changes.The main results are as follows:(1)Carbon assimilation mechanism of P.donghaiense under different concentrations of CO2 was compared.The results showed that P.donghaiense could use either HCO3-or CO2 as inorganic carbon source,but the priority was different.Under air bubbling condition with sufficient CO2,P.donghaiense preferentially used CO2 as inorganic carbon source,with a high cell density and high cell viability.Under no air bubbling condition,the concentration of seawater dissolved inorganic carbon decreased as cell biomass increased,P.donghaiense could use both CO2 and HCO3-,gene expressions and enzyme activities of CA,PEPC and PEPCK were significantly increased,meanwhile the value of 813C of particle organic carbon increased,close to that of seawater,indicating that P.donghaiense cells possesse the C4 pathway,which was highly expressed under low CO2 condition.(2)The carbon assimilation mechanism of P.donghaiense under different temperatures was compared.The results showed that P.donghaiense cells could grow in the range of 19??28?,but physiological response and carbon assimilation of cells varied significantly at different temperatures.When temperature varied among the range of 19??25? growth rate and cell viability of P.donghaiense were increased,contents of intracellular organic carbon and nitrogen were decreased,and gene expressions and enzyme activities of CA,PEPC and PEPCK were also increased,indicating the up-regulation of CCM and C4 pathway.The expression of RubisCO and its activity were decreased,which might be caused by lower turnover rate in low temperature and cells increased RubisCO expression to meet carbon demands.When the temperature increased to 28? cell growth rate of P.donghaiense maintained stable,while cell viability was significantly decreased and content of photosynthetic pigments was increased.Meanwhile,gene expressions and enzyme activities of RubisCO,CA,PEPC and PEPCK were decreased.These results showed that 25?was the optimal growth temperature for P.donghaiense.(3)The carbon assimilation mechanism of P.donghaiense under nitrogen starvation and different nitrogen sources was compared.Nitrogen starvation significantly declined cell growth rate,cell viability,and photosynthetic pigments content of P.donghaiense,meanwhile gene expressions and enzyme activities of RubisCO,CA,PEPC and PEPCK were also decreased.Three nitrogen forms,such as nitrate,ammonium and urea could be used by P.donghaiense effectively.Although no significant differences were observed in cell viability,photosynthetic pigments content and total organic carbon among three different nitrogen forms,the carbon assimilation pathway varied significantly.No significant difference in carbon assimilation pathway was observed between cells grown in NO3-and Nn4+.However,gene expressions and enzyme activities of CA,PEPC and PEPCKwere significantly decreased in the cells grown in urea,gene expression and enzyme activity of RubisCO varied insignificaly,indicating that P.donghaiense cells could use the generated CO2 from degradation of urea,and down-regulated expressions of intracellular carbon concentrating genes.