Arbuscular Mycorrhizal Fungi Alleviate Lead Stress In Medicago Truncatula

The use of arbuscular mycorrhizal fungi to form symbiositic relationship with host plant could enhance the heavy metal tolerance and improve the effect of bioremediation.However,the mechanism by which AM fungi improve heavy metal tolerance is not fully understood,which adverses to the use of AM fungi in bioremediation.In this study,the greenhouse experiment was conducted to investigate the effect of AM fungi（Rhizophagus irregularis）on reactive oxygen species（ROS）homeostasis,melatonin accumulation and regulate capacity,cell wall composition,and the metabolic capacity of carbon and nitrogen of Medicago truncatula under lead stress.This study reveals the mechanism by which AM fungi improve the lead tolerance of Medicago truncatula from both physiological and molecular aspects.The main results and conclusion as below:1. AM fungi improve ROS homeostasis of Medicago truncatula under lead stressAM inoculation improved Medicago truncatula growth condition but did not affect malondialdehyde（MDA）content and O₂.^-generate ability without Pb stress.AM inoculation decreased the accumulation of water-soluble lead in shoots and roots and dereased MDA content and O₂.^-generate ability under Pb stress.Pb stress dramatically decreased H₂O₂content and NADPH oxidase activity in AM plants.Respiratory burst oxidase homologue（Rboh）is encoding for NADPH oxidase in plants.AM inoculation upregulated the transcription of Mt Rboh C,Mt Rboh D,Mt Rboh E,Mt Rboh F and Mt Rboh G without Pb stress.Pb stress significantly downregulated Mt Rboh C,Mt Rboh D,Mt Rboh E,Mt Rboh F and Mt Rboh G expression in AM roots.The treatment of the inhibitor of NADPH oxidase（DPI）significantly enhanced arbuscule colonization and decreased antixodiant enzyme acticity in AM and non-AM roots,which indicated that NADPH oxidase may involve AM symbiosis and antoxidiant regulation.AM inoculation keeps ROS homeostasis through decreasing ROS damage,activating antioxidant response and inhibiting NADPH oxidase activity.2. Exogenous application of melatonin improves the growth and lead tolerance of mycorrhizal Medicago truncatulaAM inoculation significantly increased melatonin accumulation of Medicago truncatula under Pb stress.Acetylserotonin methyltransferase（ASMT）is the rate-limiting enzyme in melatoin synthesis.AM inoculation upregulated Mt ASMT transcription under Pb stress.The relative expression of Mt ASMT was positively related to melatonin content in roots,which indicated that AM inoculation stimulated melatonin accumulation through regulating Mt ASMT expression.Melatonin application dramatically improved biomass of Medicago truncatula,enhanced both AM colonization and Mt PT4 expression and inhibited Pb uptake in roots and shoots.Under Pb stress,melatonin application increased carotenoid content but did not affect chlorophyll a and chlorophyll b contents.Plants with melatonin application had lower MDA content and higher antioxidant activity compared with non-melatonin-applied plants.Melatonin application increased the accumulation of proline and flavone in AM and non-AM plants.These results show that the application of melatonin inhibits lead uptake,improves AM symbiosis,stimulates antioxidant capacity,and enhances the lead tolerance of mycorrhizal plants.3. AM fungi promote the accumulation of cell wall polysaccharides and cell wall peroxidase activity under lead stressAM inoculation had no significant effect on cell wall-immobilized lead content,but significantly increased the proportion of cell wall-immobilized lead.AM inoculation significantly increased the polysaccharides content of cell wall components（pectin,hemicellulose 1 and hemicellulose 2）.AM inoculation dramatically increased the polysaccharides content of cell wall under Pb stress,but AM inoculation did not affect uronic acid.ClassⅢperoxidase is encoding for cell wall peroxidase in plants.Pb stress significantly upregulated Mt Prx05 and Mt Prx10 expression in mycorrhizal root.Under Pb stress,AM inoculation significantly activated cell wall peroxidase activity.Cell wall peroxidase activity was significantly positively correlated with the relative expression of Mt Prx05.These results indicate that mycorrhizal plants have stronger cell wall-immobilized lead capacity under lead stress,which involve cell wall peroxidase activity and cell wall polysaccharides content.4. AM fungi alter the distribution of sucrose and amino acids in Medicago truncatulaAM inoculation increased phosphorus content,root surface and root volume under Pb stress,which indicated that AM fungi promote the potential of nutrient uptake.In carbon assimilation,AM inoculation dramatically enhanced photosynthetic and photoprotection capacity under Pb stress.AM inoculation decreased the shoot/root ratio of sucrose and increased sucrose content under Pb stress,which indicated that AM fungi induce the transport of sucrose from shoots to roots.In roots,AM fungi stimulated acid invertase activity and upregulated the expression of both Mt SUT1-1 and Mt SUT4-1,which suggest that AM fungi promote the unload and cleaving capacity in roots.In organic acid accumulation,Pb stress significantly decreased malic acid accumulation and increased tartaric acid accumulation in shoots.AM inoculation promoted minor amino acid（Cystine,leucine and isoleucine）accumulation in roots under Pb stress.These results indicate that AM inoculation improves Pb tolerance of Medicago truncatula through transporting sucrose to roots preferentially,increasing phosphorus uptake,and promoting minor amino acid accumulation in shoots.All in all,AM inoculation improve the lead tolerance of Medicago truncatula through decreasing ROS production capacity,improving ROS cleaving ability,enhancing melatonin synthesis and regulation ability,enchancing cell wall lead immobilizing ability,and regulating the distribution of carbon and nitrogen metabolites.This study provides a solid theoretical basis and new ideas for the technology of bioremediation using mycorrhizal plants.