| Plant roots-soil microbiome interaction is essential for plant health in natural and agricultural ecological environments.The high plasticity and complexity of crop root systems increase the challenge for stimulating the biological potential of beneficial host-microbe interactions.For crop species,the relationship between structural characteristics during root growth and development and the composition/diversity of specific microbial groups has not been established.As the rhizosphere signal molecules released by the roots,flavonoids play an important role on the interaction between roots and microbiome.Although systematic and detailed researches on interctions between legume roots and their rhizosphere microorganisms have been reported,the roles of flavonoids in the interaction of non-legume roots and their rhizosphere microorganisms are unclear.In this study,a naturally silenced dominant maize mutant C2-Idf(Colorless2-Inhibitor diffuse),defective in a gene encoding a chalcone synthase,with its wild type C2 was used to explore the key roles in the interaction corn roots and their rhizosphere microorganisms.As the main characteristics of the root systems,the formation of lateral roots and root hairs not only increases the absorption area of the root system to absorb water and nutrients,but also influences their rhizosphere microorganisms.In this study,root phenotyping experiments were performed using maize mutants defective in lateral root and root hair formation under the same nitrogen-poor soil conditions to wheath lateral root and root hair formation influence microbiome differentiation along the root.1)We investigated 20 phylogenetically distinct genotypes and showed that the genotypes performed differently in the field with respect to biomass production,leaf area,photosynthesis and shoot nitrogen accumulation.Among these genotypes,the inbred line 787 displayed highest leaf area,shoot biomass and nitrogen accumulation in comparison to the other genotypes,while LH93 performed poorly under the same field conditions.The three root zones(the meristematic zone and the apical part of the elongation zone(young zone),the basal part of the elongation zone and the apical part of the differentiation zone with root hairs and emerging lateral root primordia(transition zone)and the basal part of differentiation zone with root hairs and emerged lateral roots(maturity zone))were harvested for a transcriptomics experiment and the attached rhizosphere to a fungal and bacterial microbiome analysis.A principal component analysis(PCA)together with differential gene analysis illustrated a high transcriptomic similarity between transition zone and maturity zone in comparison to young zone.To confirm functional variation among these zones,differentially expressed genes were functionally classified by Gene Ontology(GO)term enrichment analyses.GO(Gene Ontology)enrichment analysis was used to classify the differentially expressed genes.In the comparison of the functions of the three regions,young zone vs transition zone and young zone vs mature zone are specifically enriched with genes related to root hair elongation,and transition zone vs mature zone and young zone vs mature zone are specifically enriched with genes related to lateral root development.By gene co-expression network analysis(WGCNA),it was identified that all maize genotypes showed specific abundance for root development.The 6 modules(M22-M27)were differentiated based on the root system(from young meristem area to mature differentiation area).M22-M27 showed a gradual decay trend from young zone to mature zone,but the enrichment of the three modules(M28-M30)shows the opposite trend.We also found that auxin signal transduction and phenylpropane biosynthesis pathways are highly interactive in growth module 26.The central gene acetyl-Co A carboxylase 1 in defense module 30 plays an important role in the interaction of secondary metabolism and microbial carbon/nitrogen metabolism.The root growth-related module 26 and the defense-related module 30 present antagonistic opposition,which indicates the balance between growth and defense in the process of root differentiation.The alpha diversity of bacterial microbial communities gradually decreased from young zone to mature zone,but no significant differences were found in the diversity of fungal microbial communities among the three regions.There are differences in the relative abundance of microorganisms in the three different developmental regions.For the rhizosphere microbiota with>0.1%abundance,Proteobacteria and Verrucomicrobia were significantly enriched in mature zone.On the contrary,Acidobacteria,Gemmatimonadetes,Chloroflexi and Nitrospirae showed a decreasing trend from young zone to mature zone.Acidobacteria always existed in three areas.Nitrospirales has a positive correlation with the growth module and Verrucomicrobiales has a negative correlation with the defense module.Compared with fungi,strong changes in bacterial community composition were observed during root development.Meanwhile,the transcriptome function of the host root development and the community structure of the rhizosphere bacterial microbiome are coordinated.In the rhizosphere,specific genes expressed in host roots interact more strongly with bacterial OTU than fungal OTU.In summary,it is clear that the expression of specific gene modules in the root zone is closely related to specific rhizosphere bacterial groups along the longitudinal root axis.By investigating rhizosphere microorganisms among genotypes,the maize inbred lines 787,which showed a unique bacterial community along the longitudinal rhizosphere and its corresponding transcriptomic module 5,displayed the highest correlation with bacterial taxa at the level of phyla and orders compared with all other maize genotypesThe gene encoding flavone synthase,FNSI2,has the highest correlation with module 5,and is expressed most strongly in 787.At the same time,the Oxalobacteriaceae were only enriched in the rhizosphere of genotype 787,which had the highest correlation with module 5.The inbred line 787 showed a strong tendency to enrich those taxa related to nitrogen metabolism and transformation(such as Lachnospiraceae and Nitrosomonadaceae).Soil sterilization and rhizosphere transplantation experiments revealed that 787 rhizosphere microorganisms have a growth-promoting effect.Metagenomic analysis found that genotype 787 was enriched with a large number of rhizosphere microbes related to biosynthesis,transportation and metabolism.Further research through 14C labeling and imaging experiments found that 787 roots released higher concentration of exudates to the rhizosphere.Inbred line 787 root secreteed more apigenin and luteolin flavonoids,which indicates the potential importance of flavonoid metabolism and its interaction with corn rhizosphere microorganisms.2)Significant differences were displayed between C2 and C2-Idf rhizosphere bacterial diversity and community composition.C2 wild type rhizosphere recruited an exceptionally high abundance of the taxon Massilia belonging to Oxalobacteraceae from the phylum Proteobacteria.The rhizosphere transplantation experimants found that the soil where C2 had grown significantly alleviated the symptoms of C2-Idf nitrogen deficiency.C2-Idf was further inoculated with 16 species of Oxalobacteraceae isolated from soils in different locations,which significantly increased C2-Idf above-ground biomass.By applying four different types of flavonoids with a concentration of 1?M in the paper roll system and the soil-pot system,we found the apigenin flavonoids only significantly promoted the accumulation of C2-Idf above-ground biomass and nitrogen in the soil system.Combined with the soil sterilization experiment,exogenous flavonoids and soil microorganisms work together to promote plant growth.Exogenous four different types of flavonoids application significantly affected the diversity and community composition of C2-Idf rhizosphere bacteria,and exogenous apigenin significantly increased the Oxalobacteriaceae enrichment of C2-Idf mutant rhizosphere.This indicates that Oxalobacteraceae bacteria may be a key factor affecting that apigenin flavonoids alleviated the symptoms of C2-Idf nitrogen deficiency.3)Maize root-deficient mutants significantly affected above-ground dry matter and nitrogen accumulation.The mutants rum1 and lrt1 with lateral root defective dispalyed the smallest biomass.Lateral root mutant lrt1 acquired more nitrogen than that of rum1in nitrogen-poor soil,although biomass production severely decreased in both lateral root mutants in comparison to the root hair mutants.There was no significant difference in biomass and nitrogen accumulation between the root hair mutant rth6 and the wild-type B73 under the condition of nitrogen deficiency.The lateral root defective mutants rum1 and lrt1attracted similar numbers of genotype-specific bacterial taxa differing from rth6 and rtcs.The taxon Oxalobacteraceae was specifically enriched in lrt1 mutant.Soil sterilization revealed dramatic repression of biomass production in lrt1,but no decrease was observed in rum1.Furthermore,In the nitrogen-poor soil,the correlation analysis showed that the lateral root density of lrt1 was correlated with the dry matter production after inoculated Oxalobacteraceae under nitrogen deficiency.In the case of nitrogen deficiency,inoculation of the Massilia significantly increased the density of lateral roots and the accumulation of dry matter and nitrogen in the shoots of the lrt1mutant.After nitrogen supplementation,the lateral root density,the dry matter and nitrogen accumulation in the shoots increased significantly.However,after inoculation with Massilia.The flavonoid test in the hydroponic system showed that the lrt1 mutant released more apigenin and luteolin flavonoids to the rhizosphere than that from the corresponding wild type.The soil 787 and C2 had grown,the lrt1 and rum1 rhizosphere transplantation experiment significantly increased lrt1 above-ground biomass and nitrogen accumulation.In summary,applying the integration of multi-omics methods(RNA sequencing,amplicon sequencing and metagenomic sequencing),we have analyzed the root development and rhizosphere microorganisms of three regions from 20 maize inbred lines with different phylogenetic relationships.The combination of correlation and WGCNA,rhizosphere transplantation and bacterial isolate inoculation with the application of several single-gene maize root mutants with obvious developmental defects,as well as 14CO2labeling and targeted metabolite analysis,reveals in low-nitrogen soils plant flavonoids drive rhizosphere-enriched oxalobacteriaceae to improve the biological mechanism of nitrogen deficiency traits in maize.This finding provides a theoretical basis for complex transboundary interactions and deepens our understanding of root developmental biology and its interaction with crop microorganisms. |
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