Supramolecular Architecture Of Phycobilisome-Thylakoid Photosynthetic Membrane In Red Algae And Its Response To Nitrogen Stress

Photosynthesis is the most important biochemical process which depends on the cooperation of a series of pigment-protein complexes embedded in photosynthetic membranes.The photosynthesis systems of eukaryotic red algae are similar to that of prokaryotic cyanobacteria in many aspects.Red algae are considered to be a key clade in the evolution of eukaryotic photosynthetic organisms,and they are very important in the research field of photosynthesis.The structures of thylakoid membranes in cyanobacteria and red algae are rather special,because the thylakoid(stromal)surface is covered by one layer of phycobilisomes(PBSs),a type of extrinsic photosynthetic antenna complexes.PBSs are large and highly structured assemblies of phycobiliproteins(PBPs)and linker proteins.They can be classified into four topological types:hemidiscoidal,hemiellipsoidal,bundle-slhaped and block-shaped.In natural environment,photosynthetic organisms,including cyanobacteria and red algae,have developed a variety of adaptive responses to cope with stressful conditions.For instance,during nitrogen starvation,the PBSs in non-diazotrophic cyanobacteria and red algae are degraded to provide nitrogen source for their survival Although nitrogen availability is one of the most important determinants that limits the growth of photosynthetic organisms,direct observations on the dynamics of supramolecular architectures of photo synthetic membranes from plants or algae in response to nitrogen stress are still lacking.Atomic force microscopy(AFM)has been proven to be a unique and powerful method for studying the supramolecular architectures of native photo synthetic membranes under near-physiological conditions.Although photosynthetic membranes from purple bacteria and higher plants have been studied in detail with AFM,few researches have been performed on the photosynthetic membranes of cyanobacteria or red algae with AFM.The three dimensional structures of hemidiscoidal PBSs have been identified.However,the detailed structural information of hemiellipsoidal PBSs in red algae is still not clear.So far,researches on supramolecular architectures of PBS-thylakoid membranes are confined to unicellular red algae Porphyridium cruentum in protoflorideophyceae.Reports about the morphology and structure of PBSs and the diversity of their arrangement on thylakoid membranes from red algae in different evolutionary status are limited.The supramolecular architecture of photosynthetic membrane is closely related to photo synthetic performance.The chlorophyll(Ch1)fluorescence kinetics has been widely used to study the photosynthesis of red algae,and their adaption to different growth conditions have been studied,such as irradiance,temperature,heavy metals,salinity,etc.However,the research on photo synthetic performance ofred algae during nutrient limitations is relatively limited.This project thesis focuses on the structure,function and regulation of photo synthetic membranes from red algae under environmental changes,by combining membrane biochemistry with AFM,transmission electron microscopy(TEM)and spectroscopy.We carried out systematic research from different aspects,and have made some progresses as follows:1.Supramolecular architecture of PBS-thylakoid membrane from P.crruentumTo study the supramolecular morphology of PBS-thylakoid membrane from red algae,intact PBS-thylakoid vesicles were separated with gentle operation,and the structural and functional integrity was detected by absorption spectra and fluorescence spectra.The supramolecular architectures of PBS-thylakoid membranes and PBSs were observed using AFM.The intact PBSs were also isolated,and their compositions were detected by electrophoresis.Our results revealed that cells were filled with PBS-thylakoid membranes and most of the membranes were parallel-arranged.The isolated PBS-thylakoid membranes maintained structural and functional integrity.Most of the PBS-thylakoid vesicles were nearly round with diameter of 0.5-8.5 ?m and thickness of 45-65 nm.On occasion,folds appeared on membranes,indicating that the spacial form of some thylakoid vesicles in cells was not in the same plane.Sometimes thylakoid vesicles with openings were observed,suggesting that connections might exist between vesicles for the convenience of material transfer.The openings might also be the damage resulting from the outflow of content in vesicles.The PBSs were densely packed on the thylakoid membranes,and most of them were randomly distributed.The PBSs exhibited a hemiellipsoidal morphology with length 58 ± 4 nm,width 40 ± 3 n,height 27 ± 2 nm(n>300),and the density of the PBSs was calculated to be(384±17)/?m2.Our results indicated that AFM is a powerful tool to investigate the morphology of thylakoid membranes and PBSs under near-physiological conditions.These results provide important clues on the supramolecular architectures of thylakoid membranes and PBSs from red algae in vivo.2.The regulation of supramolecular architecture of PBS-thylakoid membrane from P.cruentum during nitrogen starvationIn order to reveal the regulatory mechanism of supramoolecular architecture of photosynthetic membrane from red algae after nitrogen deficiency,alterations of the supramolecular architecture of thylakoid membrane from red algae P.cruentum during nitrogen starvation were characterized using AFM and other techniques.To characterize the physiology and biochemistry state of nitrogen-starved P.cruentum,the growth of cells and the Ch1 content in cells were measured,and the spectral changes of cells,isolated thylakoid membranes and PBSs were detected.The results showed that cell number increased during nitrogen starvation;but the proliferation rate was less than control.The Ch1 content in cells decreased,suggesting that the photosystem was influenced by nitrogen deficiency.The variation of spectra revealed that the content of phycobiliproteins decreased to a fairly low level,indicating that most of the PBSs in cells were degraded,and this was confirmed by the change of cell color.Ultrathin sections of cells were observed by TEM to analyze the overall change of thylakoid membranes in cells during nitrogen starvation.Thylakoid membranes were reduced significantly and lost their parallel and close configuration features.The variation of PBSs compositions was analyzed by electrophoresis.The changes of PBS-thylakoid membranes and PBSs during nitrogen starvation were observed with AFM.The round morphology of the thylakoid vesicles retained,and the diameters increased slightly along with nitrogen starvation The PBS density on the membranes decreased gradually,and at 20 days,the density ofPBSs on the membranes decreased to only 51/?m2.The sizes of PBSs gradually decreased during nitrogen starvation,and after 20 days of nitrogen starvation,their dimension decreased approximately 15%in length,23%in width and 48%in height,but the hemiellipsoidal morphology of the PBS was retained.Holes appeared on the thylakoid membranes and were distributed throughout the membranes.Along with nitrogen starvation,the quantity and size of holes increased.The thylakoid membranes without PBSs were scanned with AFM in order to confirm that the complexes missing from nitrogen-starved membranes were PBSs and further study the holes on nitrogen-starved membranes.To confirm our results,experiments on recovery upon addition of nitrogen was performed.After supplement of nitrogen,the color of the culture turned from yellow-green to dark red,and the spectral characteristics recovered.The density of PBSs on the membrane increased,indicating that the PBSs were re-synthesized.The results acquired by AFM were conducive to better understand the variation of thylakoid membranes and the degradation mechanism of PBS s under nitrogen stress.Our work directly observed the alterations in the supramolecular architectures of thylakoid membranes from a photosynthetic organism in response to nitrogen stress for the first time.3.The high resolution structure of hemiellipsoidal PBSUsing AFM with the advantages of natural and visual,the structures of PBSs on thylakoid membrane from P.cruentum were studied.The scanning probe,scanning parameters and method for samples spreading on mica were optimized and improved.The randomly distributed and parallel-arranged PBS s densely packed on membranes were scanned by AFM.Obvious ditch was in the middle of morphology of crowded PBS,dividing it into two parts along the long axis,and similarly arranged round ditches could be further observed on both sides,indicating that the structure of PBSs might be axisymmetric.The PBSs on thylakoid membranes were made to be sparse by means of three different ways:nitrogen starvation,low salt dialysis and nitrogen recovery for further heightening the resolution and acquiring more structural information.The quantity of PBSs on membranes decreased after nitrogen starvation,and taking advantage of this natural response,the high-resolution structures of PBSs on membranes after 20 days nitrogen starvation were detected.More detail structures could be observed than crowded PBSs,indicating that the sparse PBSs were beneficial for high-resolution observation.The PBSs were separated from membranes and disaggregated in the low salt buffer.The density of PBSs on membranes was reduced,and the integrity of PBSs was destroyed artificially.More structural information was observed,and more information about the supramolecular architecture of hemiellipsoidal PBS was obtained.New PBSs were synthesized during nitrogen recovery,thus PBSs in different states would be appeared on thylakoid membranes.The synthetic process of PBSs was primarily studied through the observation of high-resolution structures of PBSs during nitrogen recovery.Our results revealed that the sizes and topographies of PBSs were in homogeneity.The structures of larger-size PBSs were relatively complete.The ditches were less on the small-sized PBSs,suggesting that their structures were relatively simple,and the spherical protrusions were less,indicating that the amount of rods was fewer.Also obvious ditch in the middle of PBSs could be found,suggesting that the synthesis of PBSs might be symmetrical.We speculated that the core of hemiellipsoidal PBS was synthesized firstly,and then the rods were synthesized symmetrically along the long axis.The number and length of rods assembled gradually to form intact PBSs.In conclusion,the high-resolution imaging of sparsely arranged PBSs provided us with more structural information about hemiellipsoidal PBS from another novel perspective.4.Investigation of photosynthetic performance of P.cruentum in response to nitrogen starvation by Chl fluorescenceNitro gen is one of the most important nutrients for plants and algae to survive,and the photosynthetic ability of algae was related to the nitrogen abundance.The Chl fluorescence kinetics is closely related to photosynthesis of photosynthetic organisms.Here,the in vivo Chl fluorescence of nitrogen starved P.cruentum was analyzed including slow Chl a fluorescence kinetics,rapid light curve(RLC)and fast Chl a fluorescence kinetics to determine the effects of nitrogen deficiency on photosynthetic performance using a multi-color-pulse amplitude modulation(multi-color-PAM)fluorimeter.Due to nitrogen starvation,the photochemical efficiency of photosystem ?(PS ?)and the activity of PS ? reaction centers(RC)decreased,and photoinhibition occurred in PS ?.The photosynthetic apparatus were damaged and light energy conversion efficiency decreased,but the ability of electron transport did not decrease(ETo/TRo,REo/ETo).The water splitting system of the donor side of PS ? was seriously impacted by nitrogen deficiency leading to inactivation of oxygen-evolving complex(OEC),and it was confirmed by the positive K-band.The light transferred to PS ? declined,resulting in that a higher proportion of energy was used for photochemical reaction and thermal dissipation was reduced as shown by qP and qN.RLC demonstrated that the ability of nitrogen starved cells to tolerate and resist the high photon flux densities was weakened.VOI revealed that the time for reaching P phase increased,and the pool size of end electron acceptors of nitrogen starved cells increased.The density of active reaction centers decreased.Our report showed that the photosynthetic performance of P.cruentum was severely impacted by nitrogen deficiency.5.Supramolecular architecture of PBS-thylakoid membrane from multicellular red algae Porphyra yezoensis and Polysiphonia urceolataTo reveal the relationship between supramolecular architectures of photosynthetic membranes from different red algae,we investigated the PBS-thylakoid membranes of complicated multicellular P.yezoensis of proto florideophyceae and P.urceolata of florideophycidae at high evolutionary status based on the research of photosynthetic membrane of P.cruentum.The structural and functional integrity of PBS-thylakoid membranes was detected by absorption spectra and fluorescence spectra.The supramolecular architectures of PBS-thylakoid membranes and PBSs were observed using AFM.Our results demonstrated that the isolated PBS-thylakoid memibranes were structural and functional integrity.Two morphological types of isolated PBS-thylakoid membranes from P.yezoensi were found,the irregular membranes and round vesicles.Many holes appeared on irregular membranes,and the membranes were uneven with dimension of 1-10 ?m and thickness of 35-75 nm.Similar to that observed on vesicles of P.cruentum,few holes appeared on round vesicles.The vesicles were regularly smooth with diameter of 1 ?m and thickness of 35-65 nm.PBSs were densely and randomly packed on the thylakoid membranes.The PBSs exhibited hemiellipsoidal topography with fuzzy boundary possessing average dimensions of 52 × 40 × 18 nm(length x width x height).The morphologies of isolated PBS-thylakoid membranes from P.urceolata were irregular.Many holes appeared on membranes,and the membranes were uneven with dimension of 1-8 ?m and thickness of 35-60 nm.PBSs were densely and randomly packed on the thylakoid membranes.The hemiellipsoidal PBSs appeared on membranes with average dimensions of 49 × 34 × 23 nm(length × width × height).Some irregularly shaped structures and brick-shaped stack structures were also obseved.We suggested that except hemiellipsoidal PBSs,PBSs possessing other morphologies existed on the thylakoid membranes from P.urceolata.The supramolecular architectures of PBS-thylakoid membranes from multicellular red algae were observed directly for the first time.Our results deepen the investigation of light-harvesting complexes(LHC),providing a better understanding about the evolution of supramolecular architectures of photosynthetic membranes and the development of photosystem in red algae.This graduation thesis focuses on the supramolecular architectures of photo synthetic membranes in red algae and its response to nitrogen stress,the supramolecular architecture of hemiellipsoidal PBS and the regulation of photosynthetic performance of red algae after nitrogen starvation using AFM,TEM and PAM supported by spectroscopy and biochemistry.Our results revealed the regulatory mechanism of supramolecular architectures of photosynthetic membranes in red algae after nitrogen starvation visually in a new way,deepened the insight into the three-dimensional supramolecular architecture of hemiellipsoidal PBS,and provided relevant information about the evolution of supramolecular architectures of photosynthetic membranes and the development of photosystem in red algae.