Regulation Of Boron On The Secretion Of Extracellular Polysaccharides And Indoleacetic Acid Of Rhizobia

Rhizobia are capable of forming nodules on legume plants,which can fix nitrogen from the air and provide the nitrogen nutrient for host plants and promote the growth and development of legumes.During the modern agricultural production,the improvement of legumes productivity by rhizobia inoculation has become a common agricultural measure.However,due to the influence of host species,rhizobia species and biologic and abiotic factors,the nodulation capacity of rhizobia varies greatly,and it's difficult for plenty of rhizobium to effectively nodulate host plants and fix nitrogen,and to achieve the desired yield increase effect.Therefore,how to improve the inoculation efficiency of rhizobia has always been a hotspot in the research of nitrogen-fixing systems construction in legumes.Studies have showed that rhizobia can endogenously grow in plants,migrate to and colonize seed organs,and preferentially nodulate plant seedlings after seeds germination.However,at the time of infection and during the invasion process afterwards,rhizobia will induce plant defense response which in turn hinders their invasion.Extracellular polysaccharides and indoleacetic acid?IAA?secreted by rhizobia were reported to play important roles in this process,and they can promote rhizobia infection and invasion by weakening defense responses of host plant.The addition of exogenous boron contributed to rhizobia infection and invasion of rhizobia.At present,the migration and colonization of rhizobia in alfalfa inoculated with boron-treated rhizobia have been studied.Nevertheless,effects of boron-treated rhizobia on the secretion of extracellular polysaccharides,and IAA and cell morphological structure were not clear yet.Here,by treating Ensifer meliloti LZgn5f?gn5f??endogenous rhizobia,derived from test materials?and Ensifer meliloti 12531f?12531f??exogenous rhizobia,obtained from the China Microbiological Culture Collection Center?with different boron concentrations?0?ck?,0.05,1,5,10 and 100 mg/L?,the appropriate boron concentration was screened and used to treat the two strains to study the effect of boron on the production of extracellular polysaccharides and IAA and on the cell morphology and structure.Proteomics was performed to analyze the difference in protein expression between gn5f and 12531f after treated by boron,and to further clarify the regulatory mechanism of boron on the secretion of extracellular polysaccharide and IAA.Results are as follows that:1.The 100 mg/L and 1 mg/L boron levels contributed to the growth of gn5f and 12531f,respectively.The secretion of extracellular polysaccharides and IAA and the cell morphology and surface structure of gn5f and 12531f were respectively promoted by the two boron levels as well.2.Proteomics analysis showed that 100 mg/L boron treatment on gn5f caused the up-regulation of seven proteins and down-regulation of 47 proteins.Further functional classification divided these proteins into five categories:cellular processes?transport and catabolism?,environmental information processing?membrane transport?,genetic information processing?transcription?,metabolism?global and overview maps?,and other unknown functional proteins.Among them the known functional up-regulated proteins?NAD-dependent succinate semialdehyde dehydrogenase,acetyl-CoA synthetase,and succinate semialdehyde dehydrogenase[NADP?+?]?are all metabolic?global and overview maps?functional proteins.The 1mg/L boron treatment induced and suppressed the expression of three and four proteins on 12531f,respectively.They can be divided into four categories:cellular processes?transport and catabolism?,genetic information processing?transcription?,metabolism?global and overview maps?and unknown functional proteins.The up-regulated Uvr ABC system protein B belongs to genetic information processing?transcription?functional protein,and aspartate lyase and CTP synthase belong to metabolic?global and overview maps?functional protein.3.According to the main metabolic pathways enriched by differential expressed proteins,the mechanism of 100 mg/L boron promoting the production of extracellular polysaccharides and IAA by rhizobia gn5f is:boron promotes the up-regulation of NAD-dependent succinate-semialdehyde dehydrogenase,Acetyl-coenzyme A synthetase,succinate-semialdehyde dehydrogenase[NADP^?+?],which promote metabolic pathways related to the tricarboxylic acid cycle such as pyruvate metabolism,GABA bypass metabolism,and glutamate metabolism.Afterwards,the energy and various carbohydrates?D-galactose residue,D-glucose residue and D-glucuronic acid,etc.?provided by these metabolic pathways promoted the synthesis of extracellular polysaccharide.IAA can be synthesized by tryptophan via indole-3-acetamide?IAM?or indole-3-pyruvate under the action of related enzymes,and boron contributed to this process by improving tryptophan synthesis.The mechanism of 1 mg/L boron promoting the production of extracellular polysaccharides and IAA by rhizobia 12531f is:boron improves the production of fumaric acid,an intermediate product of the tricarboxylic acid cycle,by promoting the up-regulated expression of Aspartate ammonia-lyase and CTP synthase protein,after which certain metabolic pathways in the tricarboxylic acid cycle were induced to synthesize intermediate products?repeating units of monosaccharides?of extracellular polysaccharides and provide energy for this process.Finally,extracellular polysaccharides were synthesized with repeating units of monosaccharides under the modification of non-saccharide residues?acetyl,acetone,succinyl,etc.?.The down-regulated expression of dihydrolipoamide dehydrogenase protein inhibited the indole-3-pyruvate pathway,which indirectly promotes the formation of indole-acetamide?IAM?and in turn resulting in the conversion of IAM into IAA.