Research On The Functional Mechanism Underlying Adaption Of Cyanobacterial NdhK2 To High Light Stress

Under environmental stresses,cyanobacterial NDH-1 dependent cyclic electron transport around photosystem I(NDH-CET)can produce extra ATP,which is able to optimize the ratio of ATP to NADPH,improves photosynthesis and decreasing production of reactive oxygen species.In cyanobacteria,NDH-1 is located in the thylakoid membrane and is composed of multiple subunits.Among them,Ndh D and Ndh F subunits contain multiple copies and consequently,form different types of NDH-1 complexes in cyanobacteria.It is known that NdhK also contains two copies,NdhK1 and NdhK2.Recently,structural data indicated that NdhK1 contains an iron-sulfur cluster and participates in the electron transfer in the NDH-1 complex.Although NdhK2 has a high homology with NdhK1,the function of NdhK2 remains elusive.Previous studies indicated that under growth conditions,ndhK2 is not transcribed in the cyanobacterium wild-type Synechocystis sp.strain PCC 6803(WT),but is transcribed when the ndhK1 gene is deleted.Under this background,this thesis reveals the functional mechanism underlying adaption of NdhK2 to environmental stress.We mainly obtained the following research results:(1)We found that the transcription of ndhK2 is induced by high light using RTPCR method,indicating that NdhK2 work under high light conditions.(2)In order to reveal whether the loss of ndhK2 will affect the activity of NDHCET under high light conditions,an ndhK2 deletion mutant(?ndhK2)was firstly constructed,and then,chlorophyll fluorescence,photosynthetic parameters,and growth curve were monitored.We found that under high light conditions,deletion of ndhK2 impaired the NDH-CET activity,decreased the photosynthetic activity,and slowed down the growth rate.Together,these results suggest that NdhK2 works under high light.(3)In order to uncover the functional mechanism by which NdhK2 works under high light conditions,we analyzed the accumulation levels of NDH-1 complex in WT,?ndhK2 and ?ndhK1 under normal and high-light stress conditions using blue-native PAGE(BN-PAGE)and western blotting.Our experimental results that there was no significant difference in the amount of NDH-1L in WT,?ndhK2 and ?ndhK1 under normal conditions,but its amount in ?ndhK2 and ?ndhK1 was significantly reduced under high light conditions when compared to the WT.Therefore,NdhK2 is necessary to maintain the accumulation and stability of the NDH-1 complex under high light conditions.These results suggest that NdhK2 is redundant when compared to the function of NdhK1 in cyanobacteria.(4)In order to confirm the above hypothesis,the expression of NdhK1 in WT and?ndhK2 was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting under conditions of growth and high light.There is no difference in the expression of NdhK1 between WT and ?ndhK2 under normal conditions.However,under high light conditions,the expression of NdhK1 in WT evidently increased,while the expression of NdhK1 in ?ndhK2 obviously decreased.We thus conclude that the function of NdhK1 is redundant when compared to that of NdhK1 in cyanobacteria.In summary,this thesis first reveals the fact that cyanobacterial NdhK2 works under high light conditions and uncovers the redundant relationship between NdhK2 and NdhK1.Collectively,these research results not only increase the understanding of the functional mechanism of regulating the NDH-CET activity upon exposure of cyanobacteria cells to environmental stresses,but also provided more theoretical basis for the study of multi-copy genes.