Molecular Mechanism Of Leghemoglobin In Controlling Efficient Nodule Symbiotic Nitrogen Fixation

In human red blood cell,hemoglobin(Hb)carries oxygen and transmits it to various organs,which is indispensable for normal physiological activities.In plants,hemoglobin can be divided into two types,including non-symbiotic hemoglobin(ns Hb)and symbiotic hemoglobin(s Hb).ns Hb is widely present in various tissues of plants,whereas s Hb is mainly present in nitrogen-fixing nodules.Among them,a class of s Hb widely found in legume is named as leghemoglobins(Lbs).Rhizobium can use nitrogenase to reduce nitrogen to ammonia.However,excess oxygen will destroy the structure of the nitrogenase and reduce its ability to fix nitrogen.Lbs are located in the infected cells,combine with free oxygen and diliver it to the electron transfer chain in bacteroid and mitochondria.Although the study of leghemoglobin has a long history,there are still some unsettled questions.In our study,cryptic biological functions of Lbs,their transcriptional regulation mechanism and the source of their heme moiety are under a series of in-depth explorations.In our study,three Lbs(Lj Lb1,Lj Lb2,Lj Lb3)were successfully knocked out using CRISPR in Lotus japonicus.Phenotypic analysis of lb mutants and the complementation experiments indicate that three Lbs synergistically participated in the efficient symbiotic nitrogen fixation(SNF)with additive effect.In addition,non-symbiotic hemoglobin can not complement the SNF defect phenotype of triple mutant,while non-legume symbiotic hemoglobin can partially rescue mutant phenotype,indicating that the special function of symbiotic hemoglobin is absent from non-symbiotic hemoglobin.It also reveals the convergent evolution of legume and non-legume symbiotic hemoglobin.Furthermore,microscopic observation revealed the lytic vacuole formation,PHB accumulation and mitochondrial morphological destruction in lb mutant nodules;RNA-seq analysis reveals the differentially expressed genes in lb mutant nodules,include genes involved in carbon metabolism and nitrogen metabolism pathways,transcription factors and proteases.Furthemore,The loss of Lb leads to the accumulation of ROS and NO in the nodule infected cells,indicating that Lb may participate in maintaining the homeostasis of ROS and NO inside the nodule,in addition to binding and delivering oxygen.These findings provide good foundation for further study of Lb function and nodule senescence.As the earliest discovered nodulin gene,the transcriptional regulation mechanism of leghemoglobin has always been the focus of attention.Numbers of cis-acting elements were identifed in the Lb promoter regions.However,the specific transcription factors and transcriptional regulation mechanisms in controlling the infected cell specific expression patterns remain unclear yet.In this study,we found that the spatial and temporal expression patterns of Lj Lb1,Lj Lb2,Lj Lb3 were almost the same.The expression was at highest level in mature nodules and mainly concentrated in the infected cell.Subsequently,promoter analysis shows that the cis-acting elements that regulate the nodule-specific expression of the three Lbs are located within about 300 bp upstream of ATG.Dozens of candidate proteins have been identified by yeast one-hybrid and DNA pull down using the 300 bp DNA fragment as a bait,which may bind to the Lb promoter and regulate its transcription.These proteins will be further analyzed in subsequent studies and lay the foundation to explore the cell specific transcriptional regulation mechanism of Lb.Heme is the prosthetic group of Lb.In root nodule symbiotic system,both plastids and bacteroids have the ability to synthesize heme,but it is still unclear where the abundant heme in the nodule comes from.Our study attempts to explain the role of plant heme synthesis genes in nodule symbiosis.Among that,Glutamyl-t RNA reductase(Glu TR)catalyze the rate-limiting step of plant heme synthesis pathway.Lotus encodes three Glu TR genes(GTR1,GTR2,GTR3).GTR2 was significantly induced in the nodule infected cells,while GTR1 and GTR3 exhibited no obvious expression in the nitrogen-fixing zone.Subsequently,CRISPR knockout of GTR1,GTR2 or GTR1&2 by hairy root transformation showed no SNF defect phenotype.However,overexpression of Flu,the negative regulator of plant heme synthesis,showed obvious Fix-phenotype,e.g.,nodule color changed from red to white,shoot biomass was significantly lower than the control.These data indicates that genes in plant heme synthesis pathway may responsible for the prosthetic group synthesis of leghemoglobin.In summary,this study systematically clarifies the molecular mechanism of the synergistic roles of Lj Lb1,Lj Lb2,Lj Lb3 in sustaining efficient SNF in root nodules,reveals the biological function of Lb involved in maintaining ROS and NO homeostasis.The potential transcription factors involved in the regulation of Lb cell specific expression pattern were initially identified.Through the studies on Glu TR and Flu gene in root nodule,we have addressed the important contribution of plant heme synthesis to the total heme contents of root nodule.In a word,these results will strongly promote the research progress of Lb biological function,Lb transcriptional regulation and the molecular mechanism of efficient nitrogen fixation in mature nodules.