Physiological And Molecular Mechanisms Of Cyanobacterial Responsing And Adaptation To Iron Limitation

Although iron is abundant in the earth's crust,the currently oxidative atmosphere environment easily leads the ferrous oxidation to ferric and precipitated as Fe(OH)3 in the aquatic systems.Its solubility is still a key factor resulting in its difficulty to support phytoplankton growth in vast areas.Several in situ and bottle assay experiments had also demonstrated the importance of iron to phytoplankton production and chlorophyll concentration in one-third of the world's ocean.Many studies have found that phytoplankton also suffer from iron limitation in lakes.As a very representative phytoplankton,the function of iron in cyanobacteria has been extensively investigated.This key micronutrient is known to play a catalytic role in many biochemical reactions as a cofactor of enzymes and proteins-taking part in electron transport reactions,nitrogen assimilation,respiration,and chlorophyll synthesis,which makes the iron demand is about 10 times higher in cyanobacteria than the similarly sized non-photosynthetic bacterium.Thus,the high demand but low availability of iron drives the cyanobacterial evolution.However,in iron-deficient aquatic ecosystems,the effects of iron limitation on phytoplanktons do not appear alone.Many factors are also coexistence together with iron limitation,and act synergistically to influence the growth,photosynthesis,iron homeostasis of cyanobacteria.These factors mainly include the varying light intensity and quality,availability change of other nutrient elements(including trace metals and major elements),global climate change(warming,acidification,UV-B).Therefore,physiological and molecular mechanisms of cyanobacterial responsing and adaptation to iron limitation need further investigation.In this study,at first,we designed an experiment to reveal the acclimation of colonial Microcystis to iron limitation.Then,we further investigated the combined effects of iron limitation and other factors(light intensity,UV-B and DIC)on cyanobacteria,and their adaptation to iron limitation under these conditions.We subsequently constructed mutants to identify the functions of the iron-stress induced operon,isiABC operon,in photosynthesis and iron homeostasis.Finally,the response of the thylakoid membrane and proteins in thylakoid membrane to iron limitation was investegated,and we also identified the IsiA protein and analysed its biochemical properties.The main results are as follows:1.Capsular polysaccharides facilitate enhanced iron acquisition by the colonial cyanobacterium Microcystis.Comparations were conducted to investigate the different responsing and adaptation mechanisms between unicellular Microcystis and colonial Microcystis.Results show that,growth rates and levels of photosynthetic pigments declined to a greater extent in cultures of unicellular Microcystis than in cultures of the colonial form in response to decreasing iron concentrations,resulting in the impaired photosynthetic performance of unicellular Microcystis as compared to colonial forms as measured by variable fluorescence and photosynthetic oxygen evolution.These results indicate that the light-harvesting ability and photosynthetic capacity of colonial Microcystis were less affected by iron deficiency than the unicellular form.The carotenoid contents and nonphotochemical quenching of colonial Microcystis were less reduced than those of the unicellular form under decreasing iron concentrations,indicating that the colonial morphology enhanced photoprotection and acclimation to iron-deficient conditions.Furthermore,large amounts of iron were detected in the capsular polysaccharides(CPS)of the colonies,and more iron was found to be attached to the colonial Microcystis CPS under decreasing iron conditions as compared to unicellular cultures.These results demonstrated that colonial Microcystis can acclimate to iron deficiencies better than the unicellular form,and that CPS plays an important role in their acclimation advantage in iron-deficient waters.2.Diffcrcnt physiological responses of bloom-forming Microcystis and Pseudanabaena to different iron and light conditions.In the present study,the physiological responses of two Microcystis and one Pseudanabaena species to different iron and light conditions were investigated.Results show that under low light conditions,iron limitation resulted in a stronger decrease of the growth and photosynthesis of Pseudanabaena 1282 than the two Microcystis.Under high light conditions,the effects of iron limitation on the growth of the two Microcystis strains were not alleviated,but greatly alleviated for Pseudanabaena.These results suggest that growth and photosynthesis of Pseudanabaena 1282 are more susceptible to iron limitation than the two Microcystis when grown under low light conditions,but less susceptible to iron limitation under high light conditions.Meanwhile,the growth rate,the value of Ik of Pseudanabaena are higher,and the value of a of Pseudanabaena are lower,than those of the two Microcystis under all conditions except low light and iron-deficient conditions,Furthermore,Pseudanabaena also have more changeable total light-harvest pigment contents and additional phycobilins,which enhance its adaptation to varying light availability.Pseudanabaena also have higher NPQ and CAR content per cell volume than for Microcystis under all conditions.These results indicate that the utilization and adaptation of light,and photoprotection to high light for Pseudanabaena is more efficient than for Microcystis.Therefore,its coexistence with Microcystis colonies provides a higher efficiency in light capture and thus decreases its demand of iron.This study may help us to understand the strategy of Pseudanabaena acclimation to iron limitation.3.Different physiological and molecular response of cyanobacteria to different DIC and iron conditions.In this study,physiological and molecular responses of a long-term cultured freshwater cyanobacterium Synechocystis in different iron conditions to different DIC concentrations were studied.Results show that under iron-deficient conditions,the utilization efficiency for DIC of Synechocystis has an enormous decrease,and high DIC conditions could promote the synthesis of Chl a and the increase of the photosynthetic electron transport rate.Under iron-deficient conditions,77K IsiA chlorophyll fluorescence peak and the functional absorption cross section of PSII are higher under low DIC conditions than under high DIC conditions.Besides,under iron-deficient conditions,the affinity for DIC of Synechocystis was significantly induced,and the expression of DIC transporters were higher under high DIC concentration than under low DIC concentration.These results suggest that through the increased expression of DIC transporters induced by iron limitation under high DIC,Synechocystis enhanced its affinity of DIC and the carbon fixation to alleviate the effect of iron limitation,and thus reduced the damage caused by iron limitation in the cellular energy utilization.4.Different physiological responses of cyanobacteria to Ultraviolet-B radiation under iron-replete and iron-deficient conditions.In this study,physiological responses of four cyanobacterial strains(Microcystis and Synechococcus),which are widely distributed in freshwater or marine systems,were investigated under different UV-B radiation and iron conditions.The growth,photosynthetic pigment composition,photosynthetic activity and nonphotochemical quenching of the four cyanobacterial strains were drastically altered by enhanced UV-B radiation under iron-deficient conditions,but were less affected under iron-replete conditions.The intracellular reactive oxygen species(ROS)and iron contents increased and decreased respectively,with the increased UV-B radiation under iron-deficient conditions for both Microcystis aeruginosa FACHB 912 and Synechococcus sp.WH8102.On the contrary,intracellular ROS and iron contents of these two strains remained constant and increased respectively,with the increase of UV-B radiation under iron-replete conditions.These results indicate that iron-deficient cyanobacteria are more susceptible to enhanced UV-B radiation.Therefore,UV-B radiation probably plays an important role in influencing the primary productivity in iron-deficient aquatic systems,suggesting that their effects on the phytoplankton productivity may be underestimated in iron-deficient regions around the world.5.The cyanobacterial isiABC operon plays important roles in the iron homeostasis and photosynthesis.In this study,we demonstrated that sll0249(isiC)belongs to isiAB operon,and constructed mutants of these three genes to investigate their roles in photosynthesis and iron homeostasis.Results show that under iron-deficient conditions,compared with those of the WT,no effects on the PSI activity,PSI content and PSI supercomplex was found in the isiC cells,but the photosynthetic pigment contents,Fv/Fm,?PSII and NPQ have a significant decrease.Compared with isiC cells,isiB cells exhibited similar decresed of Fv/Fm and ?PSII,but more slowly re-oxidation of Qa,more decreased growth,photosynthetic pigments,electron transport rate and PSI content.Compared with isiB cells,isiA cells exhibited similar decreased Fv/Fm and ?PSII,but significantly slower re-oxidation of Qa,decreased growth,photosynthetic pigments,electron transport rate,PSI activity,PSI content and diffused PSI supercomplex.In addition,under iron-replete conditions,compared with WT,all of these three mutant have decreased cellular iron content,especially for the isiA cells.Furthermore,isiA cells have slower iron uptake rate under iron-replete conditions.We conclude that under iron-deficient conditions,all these three proteins participate in the protection of cell.IsiC manily protect PSII.Except substitute the ferredoxin,IsiB could also participate in the adaptation of cells to higher light intensity,and influence the PSI content.Except for increasing the size of the photosystem I antenna,IsiA protein also plays roles in the forming and stabilization of PSI supercomplex,and protection of cells from excess energy.Moreover,these proteins potentially participate in the regulation of iron homeostasis and iron uptake.6.Responses of a chlorophyll d containing cyanobacterium Acaryochloris marina to iron limitation.Here,we report that under iron-deficient conditions,growth rate,PC and APC contents,pigments ratio and layers of the thylakoid membrane of a chlorophyll d-containing cyanobacterium Acaryochloris marina varied significantly.Moreover,a chlorophyll d binding protein was induced by iron limitation in Acaryochloris marina,which leads to a blue shift for 4nm of the chlorophyll peak in vivo cell adsorption,and a newly formed 77K chlorophyll fluorescence peak at 747 nm.However,the shift direction of this fluorescence peak is totally different from IsiA in Synechocystis sp.PCC 6803 and Synechococcus sp.PCC 7942.The relative ratio of chlorophyll fluorescence peak at 747nm in chlorophyll-binding protein complexes F4-F5,F5 and F6 from sucrose density gradient centrifugation analysis are the highest.SDS-PAGE analysis shows that protein is significantly induced at 35 KDa,and further western blot and mass spectrometry identification demonstrated that this protein is a IsiA protein.