Study On The Mechanism Of Detoxification Of Cadmium By Aspergillus Sydowii

With the rapid development of industrialization,soil cadmium（Cd）pollution has become one of the serious environmental problems in China.In Cd contaminated soils,some rhizospheric microbial modify their physiology and metabolism and even their genetic material to adapt the heavy metal environment,resulting in a strong metal-resistant ability.Microorganisms grow slowly in soil and have very strict requirements on the environment,which restricts their development in soil remediation.It is important to investigate the tolerance and detoxification mechanism of rhizospheric microbial with high heavy metal concentration,which provide theoretical guidance for improving bioremediation efficiency.Microorganisms tend to store highly toxic heavy metals in vacuole,but few studies have been conducted at the microbial protoplasts and vacuoles.It is unclear whether vacuolar compartmentalization is a quiet compartmentalization process or a dynamic regulatory process that induces the transfer or depletion of related substances inside and outside the vacuole.Glutathione（GSH）is an important detoxifying substance in cells which has the function of chelating heavy metals and antioxidant detoxification,but its contribution in vacuolar compartmentalization and the process of regulation are unclear.Therefore,this study took the high Cd tolerance and high accumulation fungus Aspergillus sydowii DJ515-2 as the research material.In order to better research the regulatory mechanism of glutathione in vacuolar compartmentalization,the 2-oxothiazolidine-4-carboxylic acid（OTC;cysteine precursor）as a prodrug supported GSH synthesis and buthionine sulfoximine（BSO）as an inhibitor of GSH synthesis was be selected to conduct control experiments.We focused on the effect of modulating GSH content under Cd stress on the physiological indicators and the effect of Cd accumulation in Aspergillus.Subsequently,the subcellular separation technology and fluorescent probe technology were combined to further research the contribution of GSH for Cd stored and antioxidant defense mechanism in vacuolar compartmentalization under Cd stress.The main results are as follows:The effects of GSH on Cd tolerance and Cd accumulation in Aspergillus sydowii.were studied.The results showed that the biomass of Aspergillus sydowii.decreased continuously as the concentration of Cd in the medium increased,and increased the GSH levels could considerably alleviate the toxic effects.When Cd concentration was 120mg·L^-1in the medium,the biomass increased by 130.75%（wet biomass）under OTC treatment.Meanwhile,the Cd accumulation in Aspergillus sydowii.continued upward under different treatment of Cd in the medium.When modulating GSH content,the Cd accumulation in the Aspergillus sydowii.change accordingly,which indicated that GSH content could affect Cd accumulation in organisms（correlation coefficient 0.6716）.While OTC promoted the accumulation of Cd in Aspergillus sydowii.,Cd stress also increased Malondialdehyde（MDA）content to a certain extent,but did not cause significant damage to mycelium.When under BSO treatment,the accumulation of Cd reduced,and oxidative damage reduced accordingly.The separation methods of protoplasts and vacuoles from Aspergillus sydbowii.were optimized,and the regulation of GSH on Cd accumulation and chelation in vacuolar compartmentalization was studied.The results showed that the protoplasts of mycelium cells were released in largest numbers（1.68×10⁷）when KCl was 1.0 mol·L^-1,cellulase Onozuka R-10was 3%（w/v）,Snailase was 1%（w/v）,the Lysing enzyme was 0.1%（w/v）,and the lysis time was5h.And the vacuole yield was the largest（1.23×10⁶）when the p H was 7.5,incubation time was5min and the concentration of Triton X-100 was 0.015%.The research at protoplast and vacuole levels showed that 74.02%of Cd on average in protoplasts was stored in vacuole and the form of Cd mostly appears with combined form.The dissolved cadmium stored mostly in vacuoles with 81.57%on average,which indicated that vacuoles are the main storage sites for Cd.With the concentration of Cd in the medium increased,the dissolved Cd appear outside vacuole.When Aspergillus was cultured under OTC treatment,the dissolved Cd in protoplasts were decreased and the proportion of dissolved Cd in vacuole increased gradually,which indicated that vacuole could absorbs Cd²⁺to stabilize vital organelles outside the vacuole when GSH was abundant.When under the BSO treatment,the total Cd,combined Cd shows the corresponding upward and downward trends as expected,which also indicated that GSH played a crucial role in the chelation detoxification of heavy metals in vacuolar compartmentalization.The regulatory mechanism of GSH on the oxidative stress and antioxidant activity of Aspergillus sydowii.protoplasts and vacuoles were studied.With the concentration of Cd in the medium increased,the content of ROS in the protoplast increased significantly.When modulating GSH content,the content of ROS shows the upward and downward trends accordingly.The PCC analysis showed that the content of ROS in vacuole had a remarkable positive correlation with the content of Cd²⁺and oxidized glutathione（GSSG）,which indicated that Cd²⁺stimulated cells to produce ROS,and GSH as an antioxidant to remove then translate into GSSG.Meanwhile,the increase of GSSG observed in the cytoplasm enabled us to infer that redox reactions occur in the cytoplasm but not in the vacuole.When under OTC treatment,the increased GSH tended to be distributed in the cytoplasm to maintain homeostasis.When under BSO and 60mg/L Cd treatment,the content of GSH decreased in vacuole and increased outside the vacuole in contrast.With the increase of Cd concentration,the ratio of GSH/GSSG decreased,which indicating that there is an increasing oxidative damage in cells.It is worth noting that,the ratio of GSH/GSSG in the cytoplasm was almost higher than vacuole.When OTC and BSO was applied,there was more fluctuation in the vacuole.This further suggests that cells preferentially allocate resources to the cytoplasm to maintain stability.This research innovatively studied the regulatory mechanism of GSH in vacuolar compartmentalization at the protoplasts and vacuoles levels of fungi.The result showed that the high Cd tolerance of Aspergillus was reflected in vacuolar compartmentalization.GSH plays an essential role in the chelation storage of cadmium.The storage form of Cd mainly consists of less toxic combination of Cd.Meanwhile,GSH is a key player in antioxidative system,with a significant function in ROS scavenging.GSH/GSSG can also be a redox buffer to keep the cellular redox state in balance.Vacuole compartmentalization is a process of regulating the continuous allocation of resources inside and outside the vacuole.When glutathione is reduced,the cadmium ions drain to the cytoplasm and stimulate the cell to produce ROS,which results in more glutathione being distributed in the cytoplasm to maintain homeostasis.When glutathione is increased,more Cd²⁺are chelated in the vacuole,and the Cd uptake by vacuole of cadmium increased.In addition,it’s reasonable to speculate that the battleground of redox takes place in the cytoplasm,because we observed a more pronounced rise of GSSG in the cytoplasm than in the vacuole.These finding potentially has implications for the understanding of tolerance behavior and detoxification mechanisms of cells.In the future bioremediation of heavy metals in soil,the efficiency of soil remediation can be improved by developing organisms with high GSH production capacity.