| Blue-green algae,also known as cyanobacteria,are a class of prokaryotes that perform oxygen-evolving photosynthesis.They are widespread and diverse,and fix more than 35%of the Earth’s CO2 through their photosynthesis.It is well known that cyanobacteria grow under the stressful environment with nitrogen fluctuation,and adaptation mechanisms such as the synthesis and degradation of glycogen and cyanophycin help them resist stressful environment and perform efficient photosynthesis and carbon fixation.However,to date,the mechanisms by which cyanobacteria adapt to nitrogen fluctuation are poorly understood.Reported proteomic data indicated that the synthesis of phycobiliprotein occurs earlier than that of other photosynthetic complex components in the process of nitrogen-deficient chlorosis cyanobacteria turning green triggered by nitrogen addition.This phenomenon intrigued us:what is the function of these early-synthesized phycobiliprotein in cyanobacterial adaptation to nitrogen-fluctuation environment?In response to this question,we used cyanobacterial model species Synechocystis sp.strain PCC6803,combined omics analysis with physiological and biochemical experiments,and found a novel heme oxygenase involved in the synthesis and assembly of the early-synthesized phycobiliproteins,and pointed out the catalytic mechanism of this heme oxygenase and the dual-functional mechanisms of this heme oxygenase-dependent phycobiliprotein in adaptation to nitrogen-fluctuation environment.The main results are briefly described below:（1）By the means of western blotting,density gradient centrifugation,blue-green PAGE electrophoresis and spectroscopy,we found that in the process of nitrogen-deficient chlorosis cyanobacteria turning green triggered by nitrogen addition,phycobiliprotein synthesis occurs significantly earlier than the synthesis of both photosystem components,which is consistent with the previously reported proteomic research.Furthermore,we found that the assembly and function of phycobiliproteins also occurred earlier than that of the two photosystems.These results suggest that the inchoate synthesis and assembly of phycobiliproteins may play a key role in the adaptation of cyanobacteria to nitrogen-fluctuations environment.（2）Using methods of transcriptome,molecular biology,western blotting,density gradient centrifugation,blue-green electrophoresis,and spectroscopy,we found that deletion of the hypothetic gene nug1（nitrogen up-regulated gene 1）resulted in significant damage of the synthesis and assembly of inchoate phycobiliproteins,and hindered the nitrogen-triggered growth process from chlorosis.These results indicate that the nug1 gene is involved in the synthesis and assembly of inchoate phycobiliproteins during the re-greening process of nitrogen-deficient chlorosis cyanobacteria,and also provides a possibility for us to investigate the role of inchoate synthesized and assembled phycobiliproteins in the adaptation to nitrogen-fluctuation environment.（3）By means of bioinformatics,spectroscopy,liquid chromatography and mass spectrometry,we found that Nug1 protein contains a heme-binding domain—DUF2470.Further research found that Nug1 can bind to b-or c-type heme,but can only enzymatically react with c-type heme to generate biliverdin.These results indicate that Nug1 is a newly discovered c-type heme oxygenase in cyanobacteria,which catalyzes the conversion of c-type heme to biliverdin in the tetrapyrrole synthesis pathway,thereby participating in the synthesis and assembly of inchoate phycobiliproteins during nitrogen-induced re-greening from nitrogen-deficient chlorosis.（4）By comparing cell density,dry weight and phenotype of the wild-type and c-type heme oxygenase deletion mutantΔnug1 in the process of nitrogen-deficient chlorosis cyanobacteria turning green triggered by nitrogen upshift,we found that:during the process of cyanobacterial cells turning green after nitrogen addition,deletion of c-type heme oxygenase leads to a significant decrease in cell density and dry weight of cyanobacterial cells in low light,but has no effect under high light condition,indicating the light-harvesting function of c-type heme oxygenase-dependent phycobiliprotein.On the other hand,during the process of nitrogen-added greening to nitrogen-deficient yellowing,the deletion of c-type heme oxygenase resulted in a significant reduction in cell density and dry weight of cyanobacterial cells through synthesis and assembly of inchoate phycobiliproteins,suggesting another function another function mediated by c-type heme oxygenase-dependent phycobiliprotein—nitrogen capture or storage function.These results imply that the dual functions of c-type heme oxygenase-dependent phycobiliprotein in adaptation to nitrogen fluctuation environment.（5）By comparing the chlorophyll content of wild-type and c-type heme oxygenase deletion mutantΔnug1 in a continuous nitrogen fluctuation stress environment,we found that the chlorophyll content was significantly inhibited,even stopped by deletion of c-type heme oxygenase in cyanobacteria,indicating that the dual-functional mechanisms of c-type heme oxygenase-dependent phycobiliprotein plays a key role in the adaptation of cyanobacteria to nitrogen fluctuation stress.（6）In the process of cyanobacteria adapting to nitrogen-fluctuation environment,the dual-functional mechanisms of the c-type heme oxygenase suggested in this study and the previously reported arg Z-mediated cyanophycin mechanism have similar characteristic—synthesis and degradation of nitrogen storage substances.To test the relationship between these two mechanisms,we constructed their double mutant strain,Δnug1/arg Z.Further study found that the chlorophyll content in the double mutant strain decreased more seriously than that in their single mutants under the stress environment of continuous nitrogen-fluctuations environment.This result points to the fact that these two mechanisms are not redundant,but work together to defend against a stressful environment of nitrogen fluctuation.In conclusion,this study found a new type heme oxygenase involved in the synthesis and assembly of inchoate phycobiliproteins,and pointed out the catalytic mechanism of this novel heme oxygenase and the dual-function mechanisms of phycobiliprotein mediated by it in the adaptation of cyanobacteria to nitrogen-fluctuation environment.This dual-functional mechanism is different from the mechanism involving cyanophycin,but they work together to contribute to the adaptation to nitrogen-fluctuation environment,thereby ensuring efficient photosynthetic carbon fixation in cyanobacteria. |
