| Marine diatoms contribute as one of the dominant primary producers.Programmed cell death(PCD)mechanism plays an integral role in diatom life cycle and ecological status maintenance.Previous researches indicated that,multiple stress factors,such as nutrient limitation and virus infection,could induce diatom PCD.However,possible pathways and genes/proteins involved in PCD remain unclear.For further understanding of the genetics,proteomics,activation and regulation of diatom PCD,a combined results based on physiological observation,in vivo biochemical markers detection and whole-cell iTRAQ-based proteomic analysis were obtained in the marine centric diatom Thalassiosira pseudonana,indicating the comprehensive mechanisms associated with oxidative responses,stress adaption,cell acclimation,PCD induction and metabolic regulation under nitrogen/phosphorus stress.Our results showed that,compared to cells in nitrogen-replete condition(989.82?M NO3-),the ones cultured in nitrogen-limited condition(55.96?M NO3-)were significantly inhibited in growth rate and photosystem efficiency(Fv/Fm).Typical PCD characteristics were observed in cells cultured 4 days in nitrogen-limitation condition,which were accompanied with a series of acute biochemical reactions:notable up-regulation of ROS level and annexin V positive stained cell ratio,significantly increase of caspase activity and enhanced volume of fatty acid storage.Based on the biochemical results and significantly changed proteins from iTRAQ analysis,we indicated that nitrogen-limitation induced acute oxidative stress in T.pseudonana.Blockage in the chloroplast photosynthetic electron transport chain and damages of the respiratory chain complex ?/? led to an overwhelming ROS accumulation.The increase level of intracellular ROS would induce protein phosphorylation and the calcium-signaling-pathway-dependent PCD.Multiple cellular processes involved in nitrogen and carbon metabolism were rearranged to ensure the rapid regulation of cellular balance.The inhibition of nitrogen assimilation and protein biosynthesis and degradation may reduce cellular nitrogen utilization and energy consumption to keep cells alive.Increasing of TCA cycle key enzymes and lipid acid synthesis pathways may lead to enhanced lipid accumulation.In a word,the cooperation between these metabolic pathways maintained a C/N balance for cellular homeostasis and may be beneficial in terms of cell survival under nitrogen stress conditions.Cells suffered to phosphorus-limitation show a different response manner to nitrogen stress.Compared to cells in phosphorus-replete condition(40.8pM PO43-),the ones cultured in phosphorus-limited condition(0.59?M PO43-)were inhibited in growth rate without significantly decline in photosystem efficiency(Fv/Fm).Neither of the typical PCD characteristics and overproduction of ROS were detected based on TEM observation and biochemical detections.Proteomics analysis also indicated that the expression levels of the proteins involved in electron transport chains in the chloroplast and mitochondria were not significantly affected,which was corresponding to a lower level of ROS.Meanwhile,the transport capacity and utilization ability of dissolved organic phosphate were enhanced by significantly over expression of alkaline phosphatase(AP)and AP-like protein and up-regulated energy consumption.The proteins involved in ATP transport were also up-regulated,indicating an increased whole-cell metabolic rate.Thus,T.pseudonana showed a distinct different cellular response to phosphorus-limitation compared with the ROS-induced PCD mechanism to nitrogen stress in the same species.Based on our findings here,we infer that cellular mechanisms associated with cell fate determination and various stress response may be developed by diatom during the long evolutional history.These mechanisms enable diatom to efficiently cope with the rapid and intense fluctuations of multiple nutrient elements,and maintain a high biomass turnover ratio in complex marine environment. |
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