Microbiological Mechanism Of Endogenous H₂S From Rhizobium To Regulate Soybean Host Root Traits And Respond To Iron Deficiency Adaptability

The Loess Plateau is mainly dominated by alkaline calcareous soil,which is usually alkaline and has low available iron content,thus significantly inhibiting root growth and development.Legume-rhizobia symbiosis can adjust soil microbial community structure and function,thereby improving the soil environment（i.e.nutrients and microorganisms）,greatly enhancing plant uptake of soil nutrients and ultimately growth.Hydrogen sulfide（H₂S）,as a signaling molecule,is involved in the establishment of symbiotic relationship between legumes and rhizobia.However,how endogenous H₂S of rhizobia regulates root traits of host plants remains unclear.Therefore,we combined soil zymography to visualize the spatial distribution of enzyme activities at the entire root-soil interface.Hot spots and non-hot spots around the roots were located and compared by tiny-wet needle method to obtain enzyme kinetic parameters,and the rhizosphere microbial community structure was analyzed by high-throughput sequencing.Finally,soil zymography was combined with the identification of soil microbial community and the untargeted metabolic analysis of root exudates to explore the influence of endogenous H₂S of rhizobia on root characters and the microbial mechanism in response to iron deficiency.Main results for this study as follows:（1）The inoculation of rhizobium significantly promoted the growth of soybean.The plant height,aboveground biomass,root weight and nodule number were significantly increased,and the growth phenotypic of Q8 treatment were higher than those ofΔ3MST treatment.Compared with Control,total root length,root volume,and root surface area increased by 33.1%,23.4%and 34.1%,respectively,after inoculation with Q8 under iron deficiency.In addition,nitrogen concentrations in root and stem of soybean after inoculation with Q8 were significantly higher than those underΔ3MST treatment.After the addition of iron,the iron concentration in the roots,stems and leaves of soybean increased significantly,and the chlorophyll content and photosynthetic indexes（net photosynthetic rate,intercellular CO₂ concentration,transpiration rate,stomatal conductance）showed an increase trend.（2）The spatial distribution of enzyme activities showed that alkaline phosphatase（ALP）andβ-glucosidase（BG）activities were closely related to the root system,but were less distributed in the root zone.The activities of leucine aminopeptidase（LAP）and xylanase（XYN）are more dispersed and widely distributed in the root zone.In addition,the hotspot area of Q8 treatment account for 1.73%of the enzyme zymography,which was higher than that of Control andΔ3MST treatment.Inoculation with Q8 increased the XYN hot spots area from 2.56%to 3.91%under iron deficiency conditions.（3）Under iron deficiency condition,inoculation with Q8 significantly increased the Shannon index of rhizosphere soil,increased theα-diversity of bacteria and changed the microbial community structure.The rhizosphere bacterial co-occurrence network showed that the mean path and network diameter decreased after inoculation of Q8 andΔ3MST,indicating effective interactions between rhizobia and rhizosphere microorganisms.In addition,the number of edges,nodes and positive correlation ofΔ3MST were significantly higher than those of Control and Q8,indicating that the rhizosphere soil symbiosis ofΔ3MST treatment was stronger,which maybe caused by rhizosphere enrichment of rhizobium.（4）Scopolamine,a coumarin beneficial to rhizosphere bacteria,is significantly enriched under iron deficiency conditions,which may affect the assembly of rhizosphere microbial communities,thus contributing to the mutuality between bacteria and plants.Inoculation with Q8 affects glutathione metabolism,amino sugars and nucleotide sugars metabolism under iron deficiency,which may provide substrates and energy for H₂S synthesis.In conclusion,the endogenous H₂S of rhizobium mediated the process of symbiotic nitrogen fixation,thus affecting soybean root traits,rhizosphere microbial community composition,root secretion composition,and the changes of C,N and P cycle enzymes activities and spatial distribution.Moreover,Fe addition enhanced the absorption capacity of root system to Fe²⁺and improved the photosynthesis of soybean.Eventually,the two together promote the growth of soybeans.