Mechanism Of Cd Accumulation In Maize Cell Wall Enhanced By A Dark Septate Endophyte(Exophiala Pisciphila) Colonization

More prevailing evidence reveals that dark septate endophytes(DSEs)ubiquitously colonize the roots of diverse plant species under various environmental ecosystems at global,especially it’s ture for the plants naturally re-established in heavy metals contaminated environment,for their high DSE colonization intensity.Our previous research revealed that DSE colonization significantly enhanced Cd tolerance of maize against metal stress by the increased binding capacity of cell wall to lower the entry of heavy metals in the protoplast,but the mechanisms are still unclear.In the present study,maize B73 was targeted,and the mechanisms and vital roles for the cell wall bioaccumulation in the enhanced cadmium tolerance of maize(Zea mays,B73)by a dark septate endophyte(Exophiala pisciphila H93)colonization were assessed,compared with uninoculated maize under hydroponic cultures supplemented with 0 and 200 μm Cd stress.Firstly,the modifications in physico-chemical properties of maize cell walls and their functional groups involved in Cd accumulation and detoxification to increase the binding capacity,functioning a compartment as a sink for toxic trace metal accumulation were evaluated.Also,the physiological and molecular mechanisms of DSE colonization changed metabolism,modification,and synthesis of cell wall components under Cd stress was analyzed through measuring the cell wall associated enzymatic activities and gene expressions.In addition,the molecular mechanism of DSE colonization enhanced Cd tolerance of maize was analyzed by RNA-seq.The main results were summarized as follows:1.E.pisciphila H93 successfully cononized the roots of maize under hydroponic cultures with 0 and 200 μm Cd supplements,and the colonization intensity was much higher under Cd stress than the non-Cd stress.The biomass of plant shoots and roots of DSE-colonized maize was significantly increased in comparison to the control treatments.Meanwhile,the Cd accumulation in plant roots was also increased,and the translocation factor of Cd was decreased.2.Data from Transmission Electron Microscopy-Energy Dispersive X-ray Spectrometer showed that DSE colonization significantly changed Cd distribution and content in the different root tissue cells of maize,in contrast to the uninoculated-DSE maize.Cd content was decreased in xylem and phloem cells of DSE-inoculated maize,but increased in epidermis,cortex,and endodermis cells in comparison to the control treatments.Moreover,major Cd was immobilized in the cell walls of both inoculated-and uninoculated maize,which is therefore used as the main detoxification site to limit the injuries caused by Cd.These results showed that the altered accumulation in different root tissue cells and the increasing cellular compartmentalization in cell walls were one of strategies used by DSE-colonized plants.3.The Cd granular deposits were observed mainly in cell wall both in inoculated and uninoculated maize root cells by TEM,which account for more than 50%.Furthermore,Cd concentrations and proportion in inoculated-DSE maize roots were increased in comparison to the control treatments.On the other hand,cell wall components such as pectin,hemicellulose 1,hemicellulose 2,CW3(mainly cellulose,lignin,and protein)all had the ability of Cd adsorption,and hemicellulose 1 and CW3 were the major cell wall binding sites for Cd.Cd proportion was increased in pectin and hemicellulose 1 and decreased in CW3 of maize roots after inoculated with E.pisciphila H93.4.Hydroxyl,amine,amide,ester,carboxyl,polysaccharide chain,and sulfate/phosphate groups in cell wall compnonets were mainly involved in Cd chelation.E.pisciphila H93 colonization enhanced the chelation of polysaccharide chain and sulfate/phosphate groups in pectin,and hydroxyl/amine,carboxyl,and polysaccharide chain groups in hemicellulose 1.5.E.pisciphila H93 colonization enhanced the enzymatic activities of pectin methylesterase(PME),phenylalnine ammonialyase(PAL),peroxidase(POD),and upregulated the genes expressions of PME,xyloglucan endotransglucosylase(XET),POD,and laccase.These modifications resulted in the altered cell-wall components,and the changed groups in cell wall compnonets,mainly involved in Cd chelation,which contributed to the binding capacity of Cd by cell wall of DSE-inoculated maize.6.In comporison to the no Cd suppelments,Cd stress,significantly down-regulated the genes involved in phenylpropanoid biosynthesis and phenylalanine metabolism in maize root,regardless of inoculation with E.pisciphila H93 or not,and the genes involved in cutin,suberine and wax biosynthesis in inoculated with E.pisciphila H93.While,inoculation with E.pisciphila H93,up regulated these down-regulated genes caused by Cd stress,which involved in xyloglucan metabolic and hemicellulose metabolic process,and cell wall polysaccharide biosynthetic process.RNA-seq data revealed that the regulated gene pathway associated with cell wall metabolism would benefit to the Cd binding capacity of cell wall and resulted in the enhanced tolerance of DSE-inoculated maize against Cd stess.The above results indicated that cell wall was the primary site for Cd accumulation in maize roots,functioning a compartment as a sink for toxic trace metal accumulation and plays a significant role in Cd retention in roots of maize inoculated with DSE or not.Interestingly,E.pisciphila H93 colonization significantly increased Cd content of maize cell wall through changing the content of functional groups and genes expression in pectin and hemicellouse 1.On the othrer hand,E.pisciphila H93 colonization reduced Cd transportation to xylem and enhanced Cd accumulation in maize root cell wall through enhancing the gene expression levels involved in xylem metabolism and increasing thickness of xylem cell wall,which could limit Cd transportation to protoplast and shoot,and reduced the Cd toxicity to maize.These results suggested that the main mechanism of the enhanced Cd tolerance of maize by DSE colonization is to promote the Cd accumulation in mazie root cell wall.Additionally,RNA-seq data allow clues to draw more emphasis on the DSE-enhanced roles of carbon metabolism,nitrogen metabolism,oxidative response and defensive system in Cd tolerance of maize,which may have the potential to enhance Cd accumulation capacity in plants.