| Marine diatoms are a class of secondary endosymbiosis photosynthetic autotrophic unicellular algae origin180million years ago. Marine diatoms are the most important eukaryotic phytoplankton for carbon sequestration, contributing40%of global oceanic organic carbon production per year. The iron is the main limiting element ocean primary productivity, more than70%of blooms stimulated by mesoscale in situ Fe fertilization of Fe-limited High nutrient low chlorophyll (HNLC) waters were dominated by diatoms, indicating that diatoms persist in chronically Fe-limited environments and resume rapid growth when the limitation pressure is alleviated. The tolerance of diatoms to Fe limitation varies widely between species, while unique strategies of Fe uptake and storage confer competitive advantages to pennate diatoms; the molecular determinants of low Fe acclimation are largely unknown in centric diatoms.In this paper, physiological characteristics of cellular oxidative stress response and programmed cell death (PCD) in centric diatom Thalassiosira pseudonana CCMP1335under Fe-limited cultures were studied by microscopic observation, physiological measurement, cell slice and a variety of biochemical staining. We obtained the whole proteome expression information of T.pseudonana under Fe-limited conditions on day4by iTRAQ LC-MS/MS protein quantitative analysis. According to it, we analyzed the differentially expressive proteins between Fe-limited and Fe-replete cells and their function and regulation, also discussed the mechanism of response to short-term Fe limitation in T. pseudonana. It was corroborated with physiological results such as the detection of ROS activity, externalization of phosphatidylserine, caspase activity and dead cells. We obtained the basic characteristics of oxidative stress and programmed cell death in proteomics and ecological significance from physiological characteristics and proteomics results under Fe-limited conditions for T. pseudonana. The main results are listed below:1) The photosynthetic efficiency of PSII (Fv/Fm), growth rate and maximum cell abundances of the Fe-limited cells were much lower than those of the Fe-replete cells, but the activity of intracellular ROS was increased. Simultaneously, the cells showed several typical characteristics of higher plant PCD under Fe-limited, including externalization of phosphatidylserine, caspase activity increased, nucleus shrinks, organelles degradation and disappear, cell vacuolization, and cell membrane intact under Fe-limited culture conditions.2) The notable differences in the degree of in vivo cell staining for ROS production (oxidative stress), externalization of phosphotidylserine (morphological characteristic of the early stage of PCD), caspase activity and cell mortality (visualization of live/dead cells) between cells exposed to Fe-replete conditions and cells exposed to Fe-limited conditions on days4. It suggests that some cells were undergoing PCD due to intense oxidative stress on day4, combining of physiological and morphological results. From the iTRAQ data of day4samples, we obtained that41and86significant different expression profile were up-regulated and down-regulated on Fe-limited culture, respectively. They were mainly involved in "Posttranslational modification, protein turnover, chaperones","Energy production and conversion","Translation, ribosomal structure and biogenesis", and "Amino acid transport and metabolism" according to COG (Cluster of Orthologous Groups of proteins) functional classification.3) Further analysis of the proteomics results showed that light captured proteins were up-regulated and the non-cyclic electron transport chain related-proteins of photosynthesis system were inhibited by Fe-limited, which caused electrons accumulated in the electron transport chain and generated ROS after electrons combined with O2; meanwhile, the inhibited proteins of complex â…¢ and â…£ in respiratory chain also led the electronics combined with O2and generated ROS.4) The increase in ROS then trigged programmed cell death (PCD) in some of the Fe-limited cells. However, the expression of antioxidant proteins and the expression balance of pro-apoptotic and anti-apoptotic factors determined the survival and cell death for T. pseudonana under Fe-limited conditions.5) Assimilation of nitrogen was inhibited by Fe limited for T. pseudonana, and cells used nitrogen by recycle, such as amino conversion and urea cycle. We also found that there were several strategies to adapt to Fe-limited condition:down-regulate the non-essential Fe-containing proteins and up-regulate essential Fe-containing proteins or substitute Fe-containing proteins by Fe-free proteins.6) The results provide molecular-level insights into the three distinct strategies that are mainly employed by T. pseudonana in response to short-term Fe-limitation stress:firstly, the reduction of the cell population through PCD to ensure the survival cells could utilize the limited iron in the water, effectively; secondly, the metabolism of intracellular nitrogen and iron were regulated by Fe limitation to ensure the most basic survival of cells; finally, the increase in the expression of antioxidant and anti-PCD proteins to ensure the survival of the remaining cells. |
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