Identification And Functional Characterization Of Orange Carotenoid Proteins (OCP) In Terrestrial Cyanobacterium Nostoc Flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme or facai(Chinese)is distributed mainly in arid and semi-arid areas.In order to adapt to the harsh desiccated environment,N.flagelliforme has developed a series of complicated regulatory mechanisms,especially for photoprotection.The photoprotection mechanisms mainly include state transition and non-photochemical quenching in cyanobacteria.The orange carotenoid protein(OCP)plays an essential role in non-photochemical quenching mechanism.OCP is a novel light intensity sensor that can be converted from an orange inactive state to a red active state under high-light condition.Only activated OCP can bind to the phycobilisome and induce fluorescence quenching.Genes encoding OCP and its homologs were found in most of the sequenced cyanobacterial genomes,and could be classified as full-length OCP,N-terminal domain homologous protein(HCP)and C-terminal domain homologous protein(OCPC).The genome of terrestrial cyanobacteria usually contains multiple copies of ocp homologs.However,biochemical characteristics and biological functions of these OCPs are still unknown.In this dissertation,OCP homologs from N.flagelliforme,a terrestrial cyanobacterium,were identified and their functions were investigated by using biochemical and molecular method together with X-ray crystallography.The main results are as following:1.Identification and functional investigation of the red-proteins.The red-proteins from dried field samples of N.flagelliforme were purified using ion exchange chromatography and could be resolved into six visible red bands on the native-PAGE gel,these red bands were further analyzed by LC-MS/MS,respectively.The results showed that the red-proteins were composed of HCP1,HCP2,HCP3 and HCP6.HPLC and LC-MS results showed that only canthaxanthin was detected in the red-proteins.The singlet oxygen quenching activity of red-proteins was examined by EPR,the results showed that 10 μM of red-proteins can quench almost all of the singlet oxygen produced in the reaction.Both of the red-proteins and canthaxanthin could not be detected in the BG11 cultured N.flagelliforme.This result indicates that red-proteins were accumulated only in dried field samples,suggesting that OCP may play an important role in adaptation of N.flagelliforme to drought habitat.2.Transcriptional profiles of ocp homologs during rehydration and subsequent dehydration of N.flagelliforme.BLAST searches revealed that there are seven genes encode homologs of the OCP in the genome of N.flagelliforme,including two full-length ocp genes(ocpxl and ocpx2),four N-terminal domain hcp genes(hcp1,hcp2,hcp3 and hcp6)and one C-terminal domain ocpC gene.These genes were confirmed by RT-PCR.Transcription of each ocp genes in rehydrated(0,1,6 and 24h)and dehydrated(10%,50%and 90%water loss)N.flagellifomre were analyzed.The result showed that transcription of hcp1,hcp2 and hcp6 were significantly induced by dehydration of N.flagelliforme and decreased during rehydration.These results indicate that large amount of red-proteins accumulated in the dried cells of N.flagelliforme by increasing the expression of the hcp genes.3.The biochemical property and function of OCPs from N.flagelliforme.The gene encod ocp homologs and β-carotene ketolase(CrtW)were co-expressed in the β-carotene producing E.coli cells.Totally seven holo-OCP proteins were produced and purified.Absorption spectrum of OCPx2 showed a single peak at 502 nm,which is different from OCPs reported in other cyanobacterium.In addition,the absorption peaks of HCPs are closer to the blue light spectrum.Spectral analysis showed that OCPC was able to obtain the carotenoid from HCP1,HCP3 and HCP6,but not HCP2.In vitro pull-down assay showed a strong protein-protein interaction between HCP1 and OCPC.4.Both full length OCPs of N,flagelliforme belong to the OCPx protein family.In order to compare their function in photoprotection,we examined the photoconvertion and fluorescence-quenching activities of OCPx1 and OCPx2.OCPx1 has effective photoconversion activity and can recover to the inactive form very quickly in the dark.The green light activated OCPx1 can significantly quenching the fluorescence of phycobilisome in vitro.However,both the photoactivity and fluorescence quenching ability of OCPx2 were lower than OCPx1.These results indicate OCPx1 plays a major role in photoprotection under high light condition.5.The different properties of OCPx1 and OCPx2.Crystal structures of apo-OCPx1 and canthaxanthin-containing OCPx2 were analyzed.The structure of OCPx1 is similar to OCP1,while the conformation of OCPx2 is different from OCP1,mainly in the flexible loop regions between theα-helical structures in the N-terminal domain.These loop regions may affect the photoconversion activity of OCPx2 by regulating the conformation of the amino acids that interact with carotenoids.Since the holo-OCPx1 structure has not been obtained,the specific structural differences between OCPx1 and OCPx2 remain to be further studied.In this dissertation,the properties and functions of OCP homologs from N.flagelliforme were studied by using diverse methods.This work shed light the relationship between OCP homologs and desiccation-tolerant of terrestrial cyanobacteria.