Rhizosphere Processes And Mechanisms Of Cd Excluder In Maize Bioaugmented By Its Root-Associated Dark Septate Endophyte E.Pisciphila H93

Plants have evolved a series of extracellular and intracellular detoxification mechanisms in response to the nature selection of heavy metal contaminated stresses.Dark septate endophytes(DSE),as an important and integral component of plant roots,enhance the heavy metal tolerance of host plants via a variety of ways and approaches.In the present study,maize(Hui-Dan 4)and DSE strain Exophiala pisciphila H93 were targeted and used as host plant and inoculant,respectively.And an model of “exclusive Cd accumulation in maize-DSE association” was proposed and applied,which was screened in the previous experiments.And the rhizosphere progress and mechanism of exclusive Cd accumulation by DSE in maize roots and roots were comparatively investigated through the combined analyses of metabolomics with transcriptomics from the perspective of root exudates and heavy metal-transmembrane transporters.The main results acquired in this paper as follows:(1)The maize roots were successfully colonized by E.pisciphila H93 under 0 and150 ?M Cd treatments tested,and the 150 ?M Cd stress significantly increased the root colonization intensity of DSE.Compared with the non-inoculation treatment,H93 inoculation significantly reduced the Cd concentration in maize shoots and roots by 19.86%and 39.39%,respectively.Meanwhile,it is also significantly decreased the TF(Translocation Factor)of Cd in maize and inhibited Cd transfer from roots to shoots.Thus,H93 alleviated the phytotoxicity of its host maize and promoted the growth of maize shoots and roots by 49.88% and 82.85%,respectively.Data from the experiments of recultured maize in the sterile broth of preceding maize in the above 4 different treatments,displayed that the interaction between H93 and maize changed the characteristics of culture broth by the altered root exudates secreted by preceding maize,which was most crucial way to reinforce exclusive Cd absorption in maize roots and shoots.(2)Metabolomics analyses of root exudates indicated that there were distinct expression patterns of differentially expressed metabolites(DEM)induced by DSE between the comparisons under 150 ?M Cd treatments(i.e.Cd?DSE vs.Cd?n DSE)and under 0 u M Cd treatments(i.e.n Cd?DSE vs.n Cd?n DSE).In detail,compared with 23DEM(including 17.39% up-and 82.61% down-regulated)specifically regulated by DSE under 0 ?M Cd treatment,of the secretion of 15 DEM specifically induced by DSE under150 ?M Cd treatments,about 80% ug-regulated DEM were observed,conversely,20%DEM were down-regulated.Also,there were a similar trend for the 9 DEM nonspecifically induced by DSE in the above two comparisons.Correlation analyses between24 DEMs and Cd concentration in roots and shoot further showed that the decrease of Cd uptake in maize roots by DSE was a significantly negative correlation with the upregulation of 18 DEM and a significantly positive correlation with the down-regulation of 5 DEM,but the reduction of Cd accumulation in shoots by DSE only had a significant negative correlation with the up-regulation of 8 DEM in 150 ?M Cd treatments.KEGG enrichment analyses demonstrated that the 24 DEM induced by DSE inoculation were enriched in arachidonic acid metabolism,phenylalanine metabolism,lysine and flavonoid biosynthesis under Cd stress.The exogenous addition of DEM revealed that the Cd concentration of root was significantly reduced by 7.53% when 1.0 ?M 2-dimethylsuccinic acid was supplemented in the maize culture broth,coincidentally,the Cd concentration of roots was also decreased by 22.05% when maize root was pretreatment with 0.4 ?M 2-dimethyl-succinic acid.(3)Transcriptomic analyses of maize roots exhibited that the composition of differentially expressed genes(DEG)encoding metal ion transmembrane transporters regulated by DSE was significantly different between the comparisons under Cd treatments(i.e.Cd?DSE vs.Cd?n DSE)and under no Cd stress(i.e.n Cd?DSE vs.n Cd?n DSE).Clearly,comparing with 12 DEG(including 4 up-and 8 down-regulated)specifically regulated by DSE under 0 ?M Cd treatments,the different expression of 33 DEG specifically induced by DSE under 150 ?M Cd treatments were identified,in which the number of up-regulated DEG(25 DEG)was increased by 6.25 times but downregulated DEG was unchanged.Among these 33 DEG,62.16% belonged to ABC transporter family.GO and KEGG analyses further revealed that these DEG regulated by DSE also were enriched in the transmembrane transport process,the composition of membrane transporter domains,metal ion transmembrane transport activity in GO term and ABC transporters pathway in KEGG under Cd exposure,which suggested that they could play an important role in regulating the absorption or exclusion of heavy metals.(4)There was a close correlation between DEG and DEM induced by DSE under Cd treatment(Cd?DSE vs.Cd?n DSE).It suggest that DSE colonization may directly be involved with the regulation of DEG related to the synthesis of metabolites in multiple metabolic pathway(such as arachidonic acid metabolism,phenylalanine metabolism,lysine and flavonoid biosynthesis),and resulted in the differential secretion of root metabolites,and the altered root exduates finally changed the chemical forms of Cd in rhizosphere microenvironment.In conclusion,DSE colonization significantly changed the expression patterns of root exudation through the altered DEGs,which involved in multiple metabolic pathways,and resulted in the altered Cd chemical forms in rhizosphere microenvironment under Cd stress.Simultaneously,E.pisciphila H93 also directly regulated the absorption and exclusion of Cd via the specific expression of gene encoding metal ion transmembrane transporters under 150 u M Cd treatment.Our results revealed that the above two possible mechanisms to explain the enhanced tolerance and Cd excluder of a model of DSE-maize association under a Cd stressed condition.These achievements provided theoretical evidence and technical supports for preventing the accumulation of heavy metal contaminants in food chain via the bioaugmentation of DSE inoculation,and it's an alternative to gain crop safe production in the moderately and slightly polluted farmlands.