The Mechanism Of Arbuscular Mycorrhizal Fungi On Symbiosis And Lead Tolerance Of Platycladus Orientalis

Heavy metal pollution is becoming more and more serious,which seriously affects human health,environment and ecological balance.The combined application of arbuscular mycorrhizal fungi（AMF）and plants in soil remediation of heavy metal pollution is beneficial to the stable development of contaminated areas and the improvement of ecological environment.Moreover,the combined application of AMF and plants can significantly promote the growth of host plants and improve their resistance to adversity.Thus shorten the phytoremediation cycle and improve the efficiency of phytoremediation.However,the mechanism of AMF enhancing plant heavy metal tolerance is not fully understood,which is not conducive to the development of the potential of AMF in bioremediation.In this study,we analyzed the symbiosis between AMF and Platycladus orientalis,and the mechanism of AMF improving Pb tolerance was revealed by analyzing the effects on the growth status,reactive oxygen species homeostasis and glutathione transferase gene expression of P.orientalis.The main results are as follow:1.The study of symbiosis between P.orientalis and 2 AMFIt showed that both AMF can form symbioses with P.orientalis and the colonization rate was over 80%after 2 weeks of inoculation.The intensity of mycorrhizal colonization（M%）and arbuscular abundance（a%）of the 2 AMF increased continuously from a relatively low level.The growth ranges of M%and a%of R.intraradices were 4.95%-14.95%and10.54%-6.63%;The growth range of M%and a%of F.mosseae was 4.63%-18.74%and23.38%-49.11%,which tended to be stable after 4 weeks of inoculation.The aboveground and underground biomass of P.orientalis seedlings showed no significant changes after 2,4and 6 weeks of inoculation.Inoculation of R.intraradices and F.mosseae significantly increased the phosphorus concentration of P.orientalis,which were 57.99%and 90.14%,67.44%and 121.98%,64.06%and 145.51%,respectively.2 AMF could increase the chlorophyll content of P.orientalis,and the effect of inoculation of F.mosseae was better than that of R.intraradices.The symbiotic relationship between 2 AMF and P.orientalis was confirmed for the first time.In a short inoculation time（6 weeks）,both AMF improved phosphorus nutrition and promoted photosynthesis of P.orientalis.It can be seen that F.mosseae and P.orientalis had better adaptability.2.Study on the mechanism of AMF enhancing Pb tolerance of P.orientalisIt showed that Pb stress significantly reduced M%and a%.Inoculation of AMF could promote the growth of P.orientalis,enhance the fixation of Pb,and reduce the Pb translation factors（TF）.Meanwhile,Inoculation of AMF could significantly improve the root activity,total chlorophyll content and shoots and roots phosphorus content of P.orientalis under Pb stress,and improve the growth status of P.orientalis under Pb stress to a certain extent.The root activity,total chlorophyll content,shoots and roots phosphorus concentration of R.intraradices plants increased 21.79%-27.06%,44.91%-54.41%,108.40%-159.60%,38.35%-84.24%compared with CK treatment under Pb stress,respectively.On the contrary,the root activity,total chlorophyll content,shoots and roots phosphorus concentration of F.mosseae plants increased by 14.15%-24.64%,31.59%-66.44%,128.91%-218.74%and49.02%-87.99%compared with CK treatment under Pb stress,respectively.Furthermore,inoculation of AMF was beneficial to improve the activities of superoxidedismutase（SOD）,catalase（CAT）,peroxidases（POD）and glutathione transferase（GST）and the efficiency of As A-GSH cycle in the antioxidant enzyme system of P.orientalis.The H₂O₂ and MDA contents of R.intraradices plants decreased compared with CK treatment under Pb stress,17.71%-32.45%and 19.71%-22.11%in shoots,6.54%-15.38%and 8.95-28.11%in roots,respectively,while the H₂O₂ and MDA contents of F.mosseae plants declined under Pb stress,20.66%-37.49%and 15.03%-30.51%in shoots,16.75%-18.34%and 10.75%-28.75%in roots,respectively.The results showed that inoculation of AMF was beneficial to the growth of P.orientalis under Pb stress,to the maintenance of the homeostasis of reactive oxygen species,to the reduction of the oxidative damage,to the effective reduction the toxicity of heavy metals,and enhance the Pb tolerance of P.orientalis.3.Cloning,bioinformatics analysis and expression analysis of the glutathione transferase（GST）gene of P.orientalisIt showed that two GST family genes PoGST1 and PoGST2 were cloned.Sequence analysis showed that the open reading frame（ORF）of PoGST1 was 696 bp,encoding 231amino acids,predicted molecular weight was 56.7 k Da and the isoelectric point was 5.17;The ORF of PoGST2 was 672 bp,encoding 223 amino acids,predicted molecular weight was 55.2 k Da and the isoelectric point was 5.17.q RT-PCR（quantitative real-time,PCR）results showed that PoGST1 and PoGST2 expression in the roots of P.orientalis were significantly down regulated under Pb stress,while R.intraradices and F.mosseae up-regulated the expression of PoGST1 and PoGST2 in response to Pb stress.The expression of PoGST1 and PoGST2 under 500,1000 and 2000 mg×kg^-1 Pb addition level were higher than CK treatmen,indicating that AMF inoculation under a certain concentration of Pb stress can increase the expression of PoGST1 and PoGST2.In conclusion,the symbiosis between AMF and P.orientalis was beneficial to plants growth,enhanced the fixation of heavy metal Pb by roots and reduced the TF of Pb.At the same time,AMF inoculation could alleviated root damage,improved nutrient status and photosynthesis to a certain extent.In addition,AMF could eliminated reactive oxygen species in plant cells,reduce oxidative damage and effectively reduce heavy metal toxicity by improving the transport efficiency of ASA-GSH cycle and improving the activity of key enzymes（GST）in the cycle.This study provides a theoretical basis for AMF application in seedling and afforestation,ecological restoration and sustainable forestry production of P.orientalis in Pb polluted areas.