Resistance Mechanism Of Agaricus Bisporus To Cadmium Stress In Soil

The pollution of heavy metals in soil has become a major concern in the world.It is unfortunate that there are presently some drawbacks in physical,chemical and bioremediation technologies,which limit their application.Fortunately,some macrofungi have a strong ability of enrichment and tolerance to heavy metals in soil.The remediation of heavy metals in soil by macrofungi has been considered to be a promising remediation method.One of the inquisitive spots is to reveal its mechanism of resistance to heavy metals.However,most studies on the resistance of macrofungi to heavy metals focused on mycorrhizal fungi or mycelium.Cadmium(Cd)is one of the most toxic heavy metals,which is also accounted to be the primary soil heavy metal pollutant in China.Therefore,In this study,Agaricus bisporus(A.bisporus),a macrofungi,was used as material,and treated by Cd stress in soil/culture medium,followed by the physiological and biochemical test,adsorption test,transcriptomics and metabonomics analysis,to assess the distribution and adsorption characteristics of Cd,the changes in physiological,transcription and metabolism level,in hope to find the resistance mechanism of A.bisporus to Cd stress from multi-level and multi aspect.The main results and conclusions of this study were obtained as follow:(1)Cd stress in soil/culture medium had toxic effect on A.bisporus,which can inhibit the growth of mycelium and fruiting body,thus affect the development of fruiting body and lead to cell deformation.However,A.bisporus can tolerate up to414.28 mg/kg dry weight(DW)of soil Cd pollution,and the highest accumulated Cd content was 18.38 mg/kg DW,indicating that A.bisporus has strong resistance to Cd stress in soil and has the potential to repair Cd contaminated soil.In the fruiting body,the Cd contents in the cap were higher than that in the stipe.In the cap,Cd mainly existed in the hydrochloric acid extraction state(92.50-97.44%)with low-toxicity.It was mainly stored in the cell soluble fractions(54.52-67.53%)and the cell wall(20.45-29.07%),indicating that the two fractions were crucial parts for Cd compartmental detoxification.(2)The contents of malondialdehyde in the fruiting body of A.bisporus increased significantly with the accumulation of Cd.The contents of soluble sugar,protein and free proline increased significantly in the late growth period and in high Cd stress.In the fruiting body of Cd stress,the activities of peroxidase(POD)and ascorbate peroxidase(APX)were higher than those of the control group in closed-cap and cap stages,while the catalase(CAT)was higher than that of the control group in flat stage.The results showed that the accumulation of Cd in A.bisporus resulted in membrane lipid peroxidation,which led to the imbalance of cell permeability and activated the system of antioxidant enzymes.The A.bisporus increased the contents of osmoregulation substances in vivo to maintain the osmotic balance and alleviated oxidative damage by increasing the activities of CAT,POD and APX.The enhancements of osmoregulation and antioxidant defense are important physiological mechanisms for the tolerance of A.bisporus to Cd stress.(3)The A.bisporus mycelium could enrich with Cd from culture medium,which led to the change of low molecular weight organic acid metabolism.The A.bisporus mycelium mainly secreted malic acid,citric acid and tartaric acid in the early stage of Cd stress,and secreted oxalic acid,fumaric acid,formic acid and succinic acid in the later stage of Cd stress.Citric acid and malonic acid were mainly involved in the detoxification of Cd stress in the early stage of the fruiting body,while oxalic acid,succinic acid and formic acid were mainly involved in the later stage.Citric acid and oxalic acid played an important role in the extracellular,whereas malonic acid and malic acid played an important role in the intracellular of A.bisporus.The detoxification of organic acids is an important mechanisms of heavy metal tolerance in A.bisporus.(4)The mycelium,extracellular polymers(EPS)and cell wall of A.bisporus can adsorb Cd²⁺in solution.The adsorption processes were found to follow a pseudo-second-order rate equation.The Langmuir adsorption isotherm was applicable to fit the adsorption process of Cd²⁺by EPS.The Freundlich adsorption isotherm was applicable to fit the adsorption process of Cd²⁺by mycelium and cell wall.The maximum adsorption capacities of mycelium,EPS and cell wall were 17.89?583.07?48.46mg/g DW respectively.EPS and cell wall can absorb a large amount of Cd²⁺,which indicated that they were played an important role in the extracellular detoxification of A.bisporus.The adsorption of extracellular substances to Cd is the important mechanisms for A.bisporus to resist Cd stress.(5)The results of transcriptome analysis showed that:Cd stress in soil had a significant effect on the gene expression of A.bisporus fruiting body,and a total of 436differentially expressed genes(DEGs)were identified,207 of which were up-regulated and 229 were down regulated..The expression levels of genes involved in ROS production and coding of fungal hydrophobic protein,pectinase and lysozyme were significantly up regulated,whereas the expression levels of genes related to DNA polymerase,DNA repair and replication were significantly down regulated,indicating that Cd stress in soil caused cell oxidative damage,cell wall destruction and interference with DNA replication.The expression levels of genes encoding chitin synthase,glutathione S-transferase,Oligopeptide transmembrane transporter activity,ABC transporter and HSP70 were significantly up-regulated.The expression levels of genes involved in polysaccharide biosynthesis of fungal cell wall,organic acid transmembrane transport and exocrine secretion,DNA damage repair and mitosis were also significantly up regulated.On the other hand,the expression levels of genes involved in the transport of metal cations in plasma membrane were significantly down regulated.In sum,those results demonstrated that:A.bisporus could reduce the absorption of Cd by Renewing cell wall,enhancing the extracellular chelation and inhibiting the transmembrane transport of heavy metal ions.Moreover,it could reduce the toxic effect of Cd in cell by enhancing the chelation,discharging and transporting of Cd to vacuole,improving the cell's antioxidant capacity,DNA repairment and biosynthesis.(6)The results of metabonomics showed that:Cd stress in soil could significantly affect the types and contents of metabolites in the fruiting body of A.bisporus,and 82different metabolites were identified,52 of which were up-regulated and 30 were down regulated,mainly including amino acids,organic acids,lipids and nucleotides.To improve the resistance to Cd stress in soil,A.bisporus may change tricarboxylic acid cycle and glycolysis pathways,enhance the metabolism of cell membrane and cell division activity,and promote the synthesis of Cd chelating substances and phenolic substances though affecting related substances involved in metabolic pathways of amino acids,nucleotides,polysaccharides,lipids,organic acids,alkaloids,phenols and so on.Combined with the analysis of differential metabolite and DEGs,the expression patterns of 76 differential metabolites were consistent with DEGs,indicating that the changes of differential metabolites were positively regulated by differential genes.