Comparative Proteomics And Targeted Metabolomics Analysis Of Pyropia Haitanensis In Response To Salt Stress

Pyropia haitanensis grows in the intertidal zone,and periodically undergoes environmental changes such as high-tide rehydration and low-tide water loss along with tidal changes.Under the condition of dry out and water loss,algae often faced hypersaline or hyposaline stress due to water evaporation or heavy rain weather.However,after high tide rehydration,P.haitanensis could still return to the normal physiological state and showed strong salt tolerance.Therefore,P.haitanensis has become an ideal material for studying the stress-resistant physiology of algae and the salt-tolerant mechanism of plants.In previous studies,researchers have conducted in-depth exploration on the response mechanism in establishing K~+/Na~+balance,maintaining osmotic pressure balance and ROS removal of P.haitanensis under hypersaline and hyposaline stress.However,the above-mentioned response mechanisms cannot be carried out without the exchange of substances,the participation of proteins and the support of energy metabolism.Nevertheless,the response mechanism of how P.haitanensis maintains the membrane system homeostasis,protein synthesis and energy supply in response to salt stress is not clear.Therefore,in this study,the thallus Z-61 strain of P.haitanensis was used to perform proteomics sequencing analysis on algae under different hypersaline and hyposaline stress using proteomics technology,and the proteomics data were verified by combining Multiple reaction monitoring technology of mass spectrum and targeted metabolomics,to finally clarify the response mechanism of P.haitanensis to maintain the internal environment stability and energy supply under salt stress,and to explain the reasons for the differences in tolerance under different degrees of salinity treatment.At the same time,the similarities and differences of molecular response mechanisms of P.haitanensis under hypersaline and hyposaline stress were compared and analyzed,which provided data support for elucidating the molecular mechanism of salt tolerance of P.haitanensis and the cultivation of salt-tolerant varieties.The main results are as follows:(1)Under hypersaline stress,the up-regulated expression of TIM in P.haitanensis facilitates the conversion of DHAP to GAP to maintain the EMP.In addition,the enhancement of PPP may also provide EMP with an adequate intermediate and thus an adequate substrate for the TCA cycle.The EMP,the PPP and the TCA cycle can provide a sufficient carbon chain backbone for the synthesis of amino acids in P.haitanensis;At the same time,the enhanced nitrogen assimilation rate also guaranteed the synthesis of amino acids and proteins under stress.Under the condition that the thallus enhanced its protein synthesis,the up-regulation expression of recognition proteins and transporters in the endoplasmic reticulum provided a guarantee for its protein synthesis under salt stress,while the up-regulation of heat shock proteins and proteins related to the ubiquitination degradation system maintained the dynamic balance of protein folding and elimination in the thallus,and ensured the protein synthesis of the thallus under hypersaline stress.On the other hand,thallus can also increase the synthesis of fatty acids to regulate the composition and proportion of fatty acids in the organisms to enhance the mobility of membrane lipids to ensure the performance of the membrane system.Therefore,adequate energy supply,material basis and stable membrane system maintenance ensured the physiological and biochemical reactions of P.haitanensis under hypersaline stress.Moreover,compared with the HSS 110 treatment,the thallus of P.haitanensis under the HSS 100 treatment had more adequate energy metabolism,more effective protein processing and synthesis capacity,and a more stable membrane system.(2)Under hyposaline stress,P.haitanensis can avoid producing or accumulating excessive misfolded and unfolded proteins by inhibiting the synthesis of self protein,thereby reducing the harm caused by endoplasmic reticulum stress;Besides,the endoplasmic reticulum quality monitoring system and the endoplasmic reticulum-related degradation system helped the proteins to fold or remove the misfolded and denatured proteins,so as to maintain the dynamic balance of protein folding and removal in the algae.At the same time,the thallus actively increase the synthesis of fatty acids in the organisms to maintain the integrity and mobility of the cell membrane.In addition,the enhancement of EMP also provided sufficient energy for the physiological and biochemical reactions of the thallus and sufficient substrate for the synthesis of fatty acids in the thallus under stress.However,compared with the LSS 0 treatment,P.haitanensis might have more adequate energy supply,a more stable endoplasmic reticulum environment and a more complete membrane system suitable for the LSS 5 treatment.(3)By comparing the response mechanisms of P.haitanensis to hypersaline and hyposaline stress,it is found that maintaining the steady state of endoplasmic reticulum and generating sufficient energy are very important for P.haitanensis to cope with salt stress.Under hypersaline stress,P.haitanensis can promote the synthesis of amino acids and proteins and enhance the fluidity of membrane lipids,and provide sufficient energy for the organisms salt-resistant reaction by promoting the circulation of TCA cycle.Different from the response mechanism of hypersaline stress,under hyposaline stress,thallus can inhibit the synthesis of its own protein,maintain the fluidity and stability of membrane lipid,and enhance the EMP to provide energy for its own stress response.To sum up,P.haitanensis can actively respond to hypersaline and hyposaline stress through energy metabolism pathway,protein synthesis and processing,and fatty acid metabolism pathway,providing material basis and energy supply for thallus response under stress.Moreover,there are significant differences between the responses of P.haitanensis to hypersaline stress and hyposaline stress.This study provides a new vision and theoretical basis for further elucidating the salt tolerance mechanism of P.haitanensis.