Study On The Mechanism Of Response To Hyposalinity Mediated By Ascorbate-Glutathione Cycle In Bangia Fuscopurpurea(Bangiales,Rhodophyta)

Coastal ecosystems worldwide are facing intense and diverse pressures caused by global climate change.Global warming will increase glaciers melting and rainfall that leads to changes in seawater salinity.Desalination causes abiotic stress to marine organisms.In particular,intertidal seaweeds,which are alternately immersed in seawater and exposed to air due to periodic tide rhythms,are susceptible to severe hypersaline and hyposaline stresses.Bangia fuscopurpurea is a promising commercial intertidal seaweed and little is known about the mechanisms by which this species adapts to hypersaline stress.In this paper we investigated the response of key metabolites and antioxidant enzymes in the ascorbate glutathione(AsA-GSH)cycle and changes in photosynthetic physiology of the algae under hyposaline stress,analysing changes in the expression of the ascorbate peroxidase(APX)gene,which has the most significant increase in activity,isolated the c DNA encoding APX and analysed the structural properties and phylogeny of the APX protein in B.fuscopurpurea,and used the yeast two-hybrid technique to preliminarily screen for APX reciprocal proteins.Studies on the response of key metabolites and enzymes in the AsA-GSH cycle of B.fuscopurpurea under hyposaline stress revealed that under hyposalinities(15 and 0psu),the contents of reduced glutathione(GSH),total glutathione(GSH+oxidized glutathione,GSSG),cysteine(Cys),and the ratios GSH/GSSG and ascorbic acid(AsA)/dehydroascorbic acid(DHA)were significantly up-regulated;the activities of glutathione peroxidase(GPX),ascorbic acid peroxidase(APX)and monodehydroascorbate reductase(MDHAR)increased(p<0.05),and glutathione S-transferase(GST)activities decreased;the glutathione reductase(GR)activities decreased on the 1st day and recovered on the 5th day(p>0.05).The hyposalities-enhanced GSH/GSSG was weakened(still higher than control)while the GR activities were promoted by adding exogenous GSH and L-buthionine-sulfoximine(BSO).The hyposalities-enhanced GPX activities and AsA/DHA were strengthened by exogenous GSH but weakened by BSO.The dehydroascorbate reductase(DHAR)activities had no significant changes whether there was exogenous GSH or not,while DHAR activities together with DHA contents were enhanced by BSO under all salinities.The results indicated that the AsA?GSH cycle was involved in response of B.fuscopurpurea to hyposalinity by means of increasing GSH/GSSG(through promoting GSH biosynthesis and GSSG reduction accompanied by consumption of GR)and AsA/DHA(promoting AsA regeneration through MDHAR).Studies on photosynthetic physiology of B.fuscopurpurea F_v/F_munder hyposaline stress revealed that B.fuscopurpurea could protect the photosynthetic system under hyposalinity(15 psu)through its own regulatory mechanism thus maintaining high levels of maximum quantum yield of photosystem II(),photochemical quenching(q P)and quantum yield of non-regulated energy dissipation of PSⅡ[Y(NO)],and could maintain the levels of net photosynthetic rates(P_n)and dark respiration rate(R_d).Under freshwater(0 psu)conditions F_v/F_mdecreased significantly and finally stabilised at around 0.2,accompanied by a significant increase in Y(NO)and the ability to maintain R_dlevels but significantly increase P_nlevels in B.fuscopurpurea.Exogenous GSH decreases F_v/F_m,q P and increases Y(NO),P_nand R_din B.fuscopurpurea under freshwater conditions.The effects of exogenous BSO on F_v/F_m,q P and Y(NO)were the same as those of GSH,but inhibited P_nunder freshwater conditions.Studies on the transcript levels and protein properties and structure of the APX gene in B.fuscopurpurea revealed that the expression of APX gene was significantly higher than the control under freshwater conditions.And the relative expression of APX was significantly upregulated under hyposaline stress with exogenous BSO and increased with the increase of hyposaline stress.This suggests that changes in APX gene expression levels underhyposaline stress may be a cause of changes in APX enzyme activity.The APX gene is 974 bp long and contains a 729-bp long open reading frame with up to 62%GC content.APX protein consists of 242 amino acids with 16 phosphorylation sites.The APX protein of B.fuscopurpurea was 88.43%and87.19%similar to the cytoplasmic APX proteins of Neopyropia yezoensis and Neoporphyra haitanensis,respectively,and formed a separate branch in the phylogenetic tree.A yeast c DNA library with a library capacity of 1.25*10^7 CFU,an average insert fragment greater than 1200bp and a 100%positivity rate was constructed by using yeast two-hybrid technology.The APX proteins were screened for interactions and validated by slewing.The ribosomal S17 family protein were identified as having strong interactions with APX proteins of B.fuscopurpurea,and the glutamate dehydrogenase 1a(glud1a),fructose-bisphosphate aldolase and alcoholic proteins had some interactions with APX proteins of B.fuscopurpurea.Through the analysis of interacting proteins,it is found that APX protein is widely involved in various metabolism(photosynthetic and non-photosynthetic metabolism,nitrogen compound metabolism and carbohydrate metabolism,etc.),cell composition(cytoskeleton and energy storage proteins,etc.),transcriptional regulation and signaling and other life processes.