Transcriptomic And Metabolomic Analyses Of Scrippsiella Trochoidea In Different Growth Stages And During The Formation And Germination Of Pellicle Cysts

Dinoflagellate cysts（dinocysts）are the immobile cells formed at a certain stage of their life cycle.There are two types of dinocysts,resting cysts and pellicle cysts.Pellicle cysts,also known as temporary cysts,have no mandatory dormant period.They can be the product of either asexual or sexual reproduction and are regarded as an important part of the life cycle of dinoflagellates.Pellicle cysts can survive adverse conditions,and also escape from predators.Pellicle cysts play an important role in the bloom dynamics of dinoflagellates,and also have the important biological and ecological significances.Although pellicle cysts are often observed in laboratory and natural environments and germinate to vegetative cells within a short time,the molecular mechanism of the formation and germination of pellicle cysts is still unclear.Scrippsiella trochoidea is one of worldwide nearshore bloom causative species,and also the common dominant dinoflagellate in the South China Sea.Pellicle cysts play significant important roles in the occurrence and collapse of its blooms.Transcriptomic analysis was conducted on different growth stages of S.trochoidea in this study in order to highlight differentially expressed genes and to provide the molecular mechanisms for its bloom dynamics.Meanwhile,pellicle cysts of S.trochoidea were induced under cold dark condition,the changes of transcriptome and metabolome were analyzed during the formation and germination of cysts.The purpose of this study is to reveal the molecular mechanism and metabolic regulation of the formation and germination of pellicle cysts.The key results are summarized as following:（1）Gene expression was obviously changed in four growth stages of S.trochoidea.Compared with the exponential growth phase,85 genes involved in photosynthesis,carbohydrate metabolism and oxidative phosphorylation were significantly down-regulated in the lag growth phase,indicating that metabolic activity was significantly lower in the lag growth phase.Gene expression showed similar patterns in the stable and decline growth phases,which represented as no changes in photosynthetic genes but significantly up-regulating of carbohydrate metabolism and oxidative phosphorylation genes,including genes for fructose phosphate kinase,pyruvate dehydrogenase complex,citrate synthase and mitochondrial electron transport components.The results suggested that algal cells produced amounts of energy to maintain cellular activity and to homeostasis at the late growth stages.Furthermore,several genes related to endocytosis,especially those for AP-2 and clathrin,were significantly up-regulated in the stable and decline phase,which may suggest a shift of autotrophic to mixotrophic.Therefore,the increased expression of energy metabolism and endocytosis genes may play important roles in the growth of S.trochoidea,and thus supporting the maintenance of dinoflagellate blooms.（2）Only two differentially expressed genes（DEGs）were up-regulated for vegetative cells after cold darkness for 2 hours compared with the exponential vegetative cells.With them,only one was functionally annotated as casein kinase1-like protein 11,which was involved in cell signal transduction and was continuously up-regulated at further cold darkness treatment of five hours and the pellicle cysts.After treated at cold darkness for five hours,transcriptome changed remarkably and98%of total 3,701 DEGs were up-regulated,including genes for glycolysis,TCA cycle,β-oxidation and oxidative phosphorylation.These up-regulated DEGs produce energy for algal cells to resist cold stress,and to prepare materials and energy for cyst formation.（3）Gene expression was obviously changed in pellicle cysts,and 78%of the total 3,870 DEGs were down-regulated.Photosynthesis and cell metabolism genes especially for energy production were significantly down-regulated.However genes for synthesis of storage materials such as sugars and lipids were up-regulated.The results indicated that photosynthesis and cellular metabolic activities were highly reduced in pellicle cysts,while carbohydrates were accumulated ready for dormancy and germination.Furthermore,64 genes related to signal transduction were significantly changed during the formation of pellicle cysts,within which genes encoding phospholipase C and Ca²⁺transport were significantly up-regulated.The result suggests the possible role of signal transduction in the formation of pellicle cysts induced by low temperature.（4）Most DEGs were up-regulated during the incubation relative to pellicle cysts,and 1,715 of the total 1,978 DEGs were up-regulated after incubation for five hours,and 1,900 of the total 1,997 DEGs for ten hours.Photosynthesis genes were significantly up-regulated during the germination,indicating the recovery of photosynthesis.Moreover,genes encoding cellular metabolic processes such as carbohydrate metabolism and oxidative phosphorylation were up-regulated,including those encoding key enzymes,e.g.phosphofructose kinase,pyruvate dehydrogenase complex,citrate synthase and ATPase subunitβ.The results suggested that pellicle cysts produced energy through the oxidation and decomposition of storage materials,and utilized these metabolic intermediates to synthesize enzymes and proteins for cyst germination.（5）Untargeted metabolomic analysis was conducted to understand the metabolite changes during the formation and germination of pellicle cysts.A total of 1,671 and1,794 molecular features were detected under the positive and negative ion modes,respectively,within which 301 and 172 species were tentatively labeled,respectively.Significant variations were recorded in metabolites during the formation and germination of pellicle cysts.Eighteen differential metabolites were obtained in the vegetative cells after treated at cold darkness for five hours compared to those in exponential growth phase,and 15 of these metabolites increased.The metabolomic profile changed significantly in pellicle cysts,and a total of 142 differential metabolites were detected,including 89 increasing metabolites and 53 decreasing ones.Compared to the lately formed pellicle cysts,metabolomic profiles were quite different in pellicle cysts after incubation,and 149 and 106 differential metabolites were recorded after incubation for five hours and ten hours,respectively.（6）From the results of transcriptomic and metabolomic analyses in this study,genes encoding polysaccharides degradation were significantly up-regulated during the formation of pellicle cysts induced by cold darkness,which agreed with the results of metabolomic analysis recorded by the increase of low molecular sugars and phosphocreatine,and the decrease of oleic acids.The results suggested algal cells produced energy and low molecular sugars via degradation of polysaccharides and fatty acids to endure cold stress during the formation of pellicle cysts induced by cold darkness.Genes encoding for cellulase,α-amylase,sucrose transport protein,fatty acids desaturase and amino acid synthase were significantly up-regulated in pellicle cysts.Metabolomic analysis showed that low molecular sugars,unsaturated fatty acids and amino acids remarkably increased in order to save material and energy storages for dormancy and germination.During cyst germination,genes involved in carbohydrate metabolism and oxidative phosphorylation were up-regulated,and meanwhile low molecular sugars,most of the unsaturated fatty acids and amino acids decreased,suggesting that the oxidative decomposition of storage materials would be an important material and energy basis for cyst germination.