Stress Response And Long-term Acclimation Mechanisms Involved In The Formation Of Resting Cells Of The Marine Diatom Thalassiosira Pseuodnana

Diatom,one of main phytoplankton in marine ecosystem,can survive in a variety of habitats.Diatom resting stage cells induced by several unfavorable environmental factors,which include resting spore and resting cell,enable diatoms to survive under biotic and abiotic stress conditions.Once they are exposed to favorable environment,the resting stage cells would rejuvenate and even form a bloom.Therefore,resting stage cells play a key role in diatoms survive.The resting cells of Thalassiosira pseudonana can be formed to cope with 84 days' dark and cold(4?).However,little is known about stress response and acclimation mechanism involved in the resting cells formation in T.pseudonana.In the present study,a combination of morphological observations,fluorescence staining,flow cytometry,and transcriptomic approach were employed to investigate stress response and long-term acclimation mechanisms involved in the formation of resting cells.The main results were showed as following:(1)Compared with vegetative cells,several different features of resting cells were observed based on morphological observation,fluorescence staining,and flow cytometry.The chloroplasts were found to aggregate in the center of the resting cell.A smaller size and more heavily silicified in the girdle band and the valve-girdle junction region were detected.Meanwhile,less chitin content was found in frustule in resting cells.(2)RNA-seq analysis revealed that major genes related to intracellular lipid metabolism pathway,including fatty acid synthesis,elongation and degradation,unsaturated fatty acid synthesis,and branched amino acid degradation were significantly up-regulated on first-day of darkness and cold.Meanwhile,the genes encoding aminoacyl tRNA synthetase which involved in protein synthesis,ornithine cyclodeaminase,and betaine pathway were significantly up-regulated during first-day stress response.These results suggested that,to cope with the first-day stress caused by darkness and cold,the cells of T.psuedonana may collect carbon flow to lipid synthesis for energy storage and intracellular protein synthesis would rapidly increase.Meanwhile,betaine and proline could be produced to maintain cell osmotic pressure.(3)The expression levels of the genes involved in fatty acid prolongation,degradation and synthesis of unsaturated fatty acids were still up-regulated during longterm resting cell formation,although the genes involved in the synthesis of major fatty acids were significantly down-regulated.The mRNA levels of trehalose-6-phosphatase and autophagy core genes,such as Atgl and Atg8,were also significantly up-regulated.These results indicated that,to acclimatize to long-term cold and darkness,the previously stored lipids may be used to provide energy by fatty acids processing°rading for resting cells,and substances were recycled via autophagy for maintain material metabolism,as well as trehalose which maybe accumulated to maintain the protein activity in resting cells.Moreover,the number of up-regulated Myb and bZIP transcription factors increased significantly during resting cells formation,suggesting that these two transcription factors may play an important role in resting cell formation.(4)A total of 519 genes including trehalose-6-phosphate synthase encoding gene were screened as potential specific-genes associated with resting cell.TpHOX8 and TpMyb4R2 transcriptional regulation network model was also established.These specific-genes were enriched in spliceosome and the number of genes undergoing alternative splicing was increased during resting cell formation,suggesting that alternative splicing may play a key role in resting cells formation.The findings allow us to better understand the molecular mechanism involved in resting cells formation and anti-stress regulation strategies,thereby enhancing our knowledge of the role of diatom resting cells as a seed bank or carbon pump.