Regulatory Role Of Strigent Response In Algal Responses Against Environmental Stresses

Stringent response is a complicated,rapid and global regulation system,which is widely found in bacteria,algae,higher plants and metazoan.During amino acid starvation,Escherichia coli accumulates guanosine tetraphosphate?ppGpp?,and reprograms transcriptional mode by downregulating active genes of physiological significance,and upregulating genes involved in survival and stress responses,to cope with adverse environment.As one of the representatives of cyanobacterial bloom,Microcystis aeruginosa exhibits very strong ecological adaptability,which lives in a complicated and changeable environment.M.aeruginosa seems to deal with varous environmental stresses easily,and bloom under certain stimuli.Haematococcus pluvialis is known as the best bioresource for natural astaxanthin.Under proper environmental stresses,it could massively accumulate astaxanthin,up to 4%dry cell weight.These two algae both exhibit distinct properties in physiological and ecological adaptability against environmental stresses,which might be mediated by stringent response.In this thesis,the RSH homologous genes Marsh and Hprsh in M.aeruginosa and H.pluvialis were cloned,respectively.Gene functional complementation,site-derected mutagenesis of key amino acids,and in vitro enzyme activity assays showed that both genes encode a bifunctional RSH with hydrolytic and synthetic activity.When supplied with Mg²⁺,both enzymes catalyzed the synthesis of ppGpp;while provided with Mn²⁺,both hydrolyzed ppGpp.Meanwhile,UHPLC methods for detecting cellular ppGpp and carotenoids in algae were developed,respectively.Study of the physiological and biochemical effects of environmental stress on algal cells found that hyperosmolality shut down stringent response in M.aeruginosa,which increased membrane permeability,caused leakage of cytoplasm,decreased photosynthetic efficiency of photosystem II?Fv/Fm?,and caused huge ROS.While nitrogen starvation induced the transcription of Marsh and thus triggered stringent response,which allivated the damage caused by hyperosmolality through preserving membrane integrity,maintaining high Fv/Fm,and reducing cellular ROS.Stringent response induced by nitrogene starvation inhibited and eliminated cellular ROS through:?1?upregulating the transcription of D1 protein in PSII to improve Fv/Fm,reduce the leakage of electros caused by hyperosmolality in the electron transfer chain,and decrease the level of cellular ROS produced by the leakage of electron;?2?upregulating the transcription and enzyme activities of oxidoreductases such as MaPOD,MaSOD,MaGAPX and MaGR to improve the ability to eliminate cellular ROS.Descrease in cellular ROS alleviated the damage to biomacromolecules caused by hyperosmolality,such as protein,nucleic acid and lipid,preserve membrane integrity,and thus prvent the leakage of cytoplasm.In H.pluvialis,nitrogen starvation,high light and hyperosmolality upregulated the transcription of Hprsh,and thus triggered stringent response in H.pluvialis cells.Stringent response induced by environmental stresses then led to the rapid transformation of green vegetable cells into aplanospore,meanwhile promoted massive accumulation of carotenoids by differentially upregulating key genes in the biosynthetic pathway of carotenoids,inculding Hpgpps,Hpfpps,Hpggpps,HplcyB and HpchyB.Conclusively,stringent response plays an important regulatory role in the responses against environmental stress,renders strong physiological and ecological adaptation to algal cells.