Research On The Molecular Mechanism Of Pyropia Haitanensis Under Desiccation Stress

Pyropia haitanensis is an economic macroalgae grows in the intertidal zone of south China.The thallus of P.haitanensis is subjected to undergo a process of emersion and immersion due to the periodic exposure to tidal fluctuations.They are exposed to air for a long time and lose over 90%cellular water at the low tide.When the tide is high,they are submerged in seawater and recover rapidly,indicating that P.haitanensis can tolerate extreme desiccation.However,there is no comprehensive explanation for how P.haitanensis adapts to this environmental stress.In this study,we selected the Z-61 strain of P.haitanensis as experimental materials,analysed the physiological characteristics,transcriptome,proteome and metabolome of the thallus under different dehydration gradients(0%,30%,60%,90%)and rehydration conditions.Then,we found key metabolic pathways and functional genes by integrated analysis of omics data.Finally,we identified the function of the key gene by transgenic experiments.This study systematacially explored the response of P.haitanensis to desiccation stress,preliminarily explained the molecular mechanism and provided basic data for further research.The main results are as follows:1.The antioxidant indexs of P.haitanensis under different dehydration gradients and rehydration conditions show that 60%water loss rate is an important boundary in response to desiccation stress.When the water loss rate was lower than 60%,intracellular H2O2 increased significantly,which activated the antioxidant enzyme CAT to remove.When the water loss rate was greater than 60%,intracellular O2-increased significantly,which activated SOD to convert it into H2O2.At the same time,APX and GSH were induced to maintain the intracellular redox balance.These results indicate that P.haitanensis has an efficient antioxidant system and could effectively eliminate ROS induced by desiccation stress.In addition,the increase of free proline and soluble sugar indicate that the osmotic adjustment also plays an important role in resisting desiccation stress in P.haitanensis.2.A total of 2,283 differentially expressed genes were obtained by analysing the transcriptomes of P.haitanensis under different dehydration gradients and rehydration conditions.These genes are mainly enriched in metabolic pathways such as carbon metabolism,biosynthesis of amino acids,photosynthesis,protein processing,pentose phosphate pathway and glycolysis.The expression patterns of these genes revealed the molecular response underlying the desiccation stress of P.haitanensis.During the period of desiccation,cells down-regulated photosynthesis,protein synthesis and degradation,and activated antioxidant system to reduce the accumulation of intracellular ROS and misfold proteins.Cells can also keep their shape and osmotic pressure by cell wall modification,synthesis of cytoskeleton and osmotic substances.In addition,glycolysis might be the main pathway to supply energy.3.iTRAQ technology was used to compare the proteomes of P.haitanensis under different dehydration gradients and rehydration conditions,and 303 differential expressed proteins were obtained.These proteins are mainly involved in protein synthesis and degradation,defense response,energy metabolism,cell structure,photosynthesis and carbohydrate metabolism.Analysis of the expression patterns of these proteins revealed the molecular response of P.haitanensis under the desiccation stress.Photosynthesis and protein synthesis were inhibited during the cells under the dehydrated state.The accumulation of intracellular ROS and misfolded proteins was caused by continuous desiccation stress,which activated antioxidant systems and chaperones to remove excess ROS and repair impaired proteins.The shape and osmotic pressure of cells were protected by cell wall modification,synthesis of cytoskeleton and carbohydrates.It is ineviTable that some cells were injured by desiccation and induced PCD to remove them.The glycolysis and TCA cycle provide energy for cell metabolism and prepare for rehydration.4.LC-MS was used to detect the metabolome of P.haitanensis under different dehydration gradients and rehydration conditions,and a total of 5,402 differential metabolites were obtained.Analyzing the changes in the metabolites such as osmotic substances,amino acids,antioxidants,phytohormones and arachidonic acid,the molecular response of P.haitanensis under desiccation stress was revealed.During the period of desiccation,cells accumulated a large amount of osmotic substances and antioxidants to maintain osmotic pressure and scavenge ROS.Desiccation inhibits the synthesis of proteins and accelerates the degradation of proteins,resulting in an increase of amino acids.Amino acids can participate in multiple stress-resistance pathways and provide energy and nitrogen for rehydration of cells.Some phytohormones also participate in the response of P.haitanensis to desiccation and rehydration.Arachidonic acid may participate in the process of stabilizing the cell membrane under desiccation stress.Metabolites related to arachidonic acid play an important role in the process of rehydration.5.The results of integrated analysis between transcriptome and proteome show that the most expression patterns of the differential genes and proteins were not consistent,suggesting that post-transcriptional regulation and post-translational modification exist in P.haitanensis.Twenty-seven differential expressed genes and proteins were identified.These genes and proteins are involved in pentose phosphate pathways,inositol metabolism,glycolysis,antioxidants,photosynthetic electron transport,cell wall synthesis,molecular chaperones and photosynthesis.We found that the pentose phosphate pathway played an important role in the response to desiccation of P.haitanensis.Integrated analysis of the proteome and metabolome showed that transketolase plays a key role in desiccation tolerance.6.A gene expression system of Chlamydomonas reinhardtii was constructed to verify the function of the transketolase gene.The results showed that transgenic C.reinhardtii successfully expressed the transketolase gene of P.haitanensis and improved the desiccation tolerance.In summary,the molecular mechanism of P.haitanensis in response to desiccation stress was obtained.During desiccation stress,cells can down-regulate photosynthesis and protein synthesis,activates antioxidant systems,chaperones and ubiquitin system to reduce oxidative damage and protein damage.Meanwhile,cells can also keep shape and osmotic pressure by cell wall modification,synthesis of cytoskeleton and osmotic substances under prolonged desiccation stress.It is ineviTable that some cells are injured by desiccation and induce PCD to remove them.Phytohormones,such as salicylic acid,jasmonic acid,zeatin and gibberellin,regulate the response of P.haitanensis to desiccation.Cells mainly rely on glycolysis and TCA cycles to provide energy for desiccation response.