The Physiological And Biochemical Characteristics Of The Three Diatoms Resting Stage Cells Formation

The diatoms resting stage cells are a special life stage in which diatoms formed with the increasing degree of the cell wall silicification under the environmental stresses.The resting stage cells are of great significance to the persistence of the diatom's own population and play a key role in the environment ecology.However,little is known about the physiological and biochemical characteristics of diatoms during their resting cells formation.In the present study,the physiological and biochemical characteristics and changes in the resting stage cell formation of three species of diatoms Thalassiosira pseudonana,Chaetoceros muelleri,Ditylum brightwellii were investigated,including cell morphology,photosynthetic characteristics,cell viability,absorption of nutrients,biological macromolecule content,etc.The comparative analysis of proteins in cell wall and organelle between resting stage cells and vegetative cells of the diatoms provided strong molecular biological evidence for changes in physiological and biochemical characteristics of the resting stage cells.The similarities and differences of the physiological and biochemical characteristics between three kinds of diatom resting stage cells were also compared to obtain metabolic characteristics during diatom resting stage cell induction.All the results revealed a strategy how diatoms achieve long term survival through resting stage cells process under environmental stresses.The main findings of this study are as follows:(1)Under low temperature(4?)and dark for 3 months,resting cells of T.pseudonana can be successfully induced.At week 8(day 56),a stable high proportion(77.64%)of mature resting cells was formed.The formation rate of resting cells increased to 93%at weeks 10-12.Silica wall thickening,cytoplasm condensation,and increased number of membranous organelles were observed in the resting cells of T.pseudonana under microscopy.During the formation of resting cells,Fv/Fm,continuously decreased to 0.076,while at the same time,the compensation accumulation of single cell Chlorophyll a occured,especially during the formation of mature resting cells.In the induction process of resting cells,the absorption and utilization of silicon and phosphorus were significantly increased.The absorbed silicate was mainly used for the silicic wall thickening of resting cells.The highest single cell intracellular silicon content and cell wall silicon content were approximately 2.5 and 3 times of those of vegetative cells,respectively.Absorption of phosphate was mainly used for accumulation of phosphorus-containing organic compounds in resting cells.The intracellular lipid significanlty accumulated in the resting cells during the middle period of induction,which may provide material and energy support for the storage of proteins found in the mature resting cells.Compared with vegetative cells,silicified cell wall associated protein Cingulin W1 and Silaffin2 were not detected in resting cells,suggesting that these proteins may not be involved in the thickening of the silica wall of mature resting cells.More organelles proteins such as membrane proteins,ribosomal proteins,energy-related proteins,pigment proteins,and phosphoproteins were found in resting cells,which may contribute to the accumulation of Chla,the conversion of oil to protein storage and the absorption and utilization of phosphate during the formation of resting cells,.(2)The formation of resting spores of C.muelleri was induced by co-stress factors:nitrogen and iron deficiency and high salinity stress.The formation rate of resting spores was the highest at day 24(72.92%).The resting spore formation process can be divided into three stages:early,middle and late stages,according to the characteristics of the resting spore formation.Silica wall thickening,cytoplasm condensation,increased number of membranous organelles,and prominent bulges in the upper valve were main morphological characteristics of resting spores of C.muelleri.Similar to the formation of resting cells of T.pseudonana,the single cell Chla content gradually accumulated during the overall decline of its Fv/Fm.The absorption of silicon and phosphorus was significantly increased and utilized.The highest silicon content of single cell wall in resting spores was about 4 times that of vegetative cells.Total sugars and lipid were accumulated during the early and mid-term of resting spores formation,whereas total phosphorous and protein were increased in the late term when matural resting spores formed.Copper-induced girdle band-associated cell surface protein was not detected in resting spores compared to vegetagable cells,which indicated that this protein may not participate in cell wall thickening of resting spores.More ribosomal proteins,ATP-related proteins,mitochondrial and energy-metabolizing proteins,and phosphoproteins were found in the resting spore organelle proteins,indicating that resting spores have higher activity in protein translation and energy metabolism,and could support the accumulation of phosphorus-containing organic compounds in resting spores.(3)Nitrogen limitation successfully induced the formation of resting spores of D.brightwellii.The formation rate of resting spores was highest at day 15,reaching to 63.5%.Its resting spore formation also had a similar stepwise increase process with C.muelleri.D.brightwellii resting spores contains a thickened silica wall and the cytoplasm was concentrated.Besides,multiple layers of new valves were formed in the original cells,and the old valves usually fall off and the cells became smaller when matural resting spores were formed.Totally different from T.pseudonana and C.muelleri,the Fv/Fm value increased after decreasing to 0.106 and single cell Chla content decreased at days 15-18 when higtest formation rate of resting spores was reached.The accumulation of cell wall silicon content in single cell appeared early at day 6,about three times that of vegetative cells,and the intracellular silicon content of single cells increased significantly in the middle phase of induction,about two times that of vegetative cells.In the resting spore induction process,phosphorus absorption increased and the intracellular phosphate content of single cell was significantly higher than that of vegetative cells.The lipid content increased but with a fluctuating change,indicating that the lipid was continuously synthesized and used.The accumulation of protein was significantly happened on day 6,with the content higher than that of vegetative cells.(4)The common features of the three diatom resting stage cells are as follows:thickened cell walls,concentrated cytoplasm,increased membrane plastids,increased cell viability,decreased FvWFm,accumulation of Chla(except for D.brightwellii),silicon and phosphorus absorption was significant,nitrate-nitrogen absorption was not significant,lipid and protein synthesis increased.However,the degree of change,the stage of occurrence and the storage format in these characteristics were different.