Study On The Molecular Response Of Kentucky Bluegrass To Cadmium Stress And The Mitigation Effect Of Exogenous NO

During the last decades,the excessive discharge of"three wastes from industry and mining"caused by human activities has caused serious soil heavy metal pollution,seriously endangering human health.Therefore,it has become an important environmental problem to be solved urgently.Phytoremediation is a rapidly emerging technology for removing toxic heavy metals.Compared with the traditional treatment methods,phytoremediation has the advantages of economy,environmental protection,safety and aesthetics,and is a method with great potential to treat heavy metal contaminated soil.Kentucky bluegrass is a perennial and cold-season turfgrass widely cultivated in China,with developed root system,good ability to absorb and accumulate cadmium,so it is a potential plant for remediation of soil polluted by cadmium.This study systematically evaluated the tolerance and response mechanism of different Kentucky bluegrass materials to cadmium.The biological techniques were used to study the effect of cadmium stress on the transcriptional level of Kentucky bluegrass,and the mechanism of applying exogenous NO to the tolerance of cadmium was also discussed.In this study,specific research conclusions are as follows:1.The photosynthetic pigment content,malondialdehyde（MDA）and proline（Pro）content,superoxide dismutase,peroxidase and catalase activity,dry matter content,and leaf relative water content of 10 Kentucky bluegrass materials seedlings under different concentrations of Cd(0,200,400 and 600μmol·L^-1)were determined.Using the membership function comprehensive evaluation method,the cadmium resistance of 10 materials was evaluated comprehensively,and selected"Midnight"as Cd resistance material and"Rugby2"as Cd sensitive material.2.The content of endogenous NO increased in Kentucky bluegrass when treated with 1000μmol·L^-1 Cadmium（CdCl₂·2.5H₂O）.When treated with nitric oxide synthase inhibitor,nitrate reductase inhibitor and nitric oxide scavenger respectively,the activities of antioxidant enzymes（SOD,CAT and ascorbate peroxidase）,chlorophyll a,chlorophyll b,carotenoids and malondialdehyde content in seedling leaves of Kentucky bluegrass under Cd stress were significantly increased,while peroxidase activity,free proline content,relative water content,dry matter quality were declined.The results indicated that the nitric oxide synthase pathway may be the main pathway of the synthesis of NO in Kentucky bluegrass,and endogenous NO can alleviate cadmium stress by regulating antioxidant enzyme activity,photosynthetic pigment content,MDA and free proline content.3.Compared with the 14 days of cadmium treatment with 1000μmol·L^-1,low concentration of NO(i.e.25,50,75μmol·L^-1)could alleviate the damage caused by cadmium to plants to different degrees,while high concentration of NO(i.e.250,500μmol·L^-1)had no significant effect on the alleviation of cadmium stress,that is,the exogenous NO had a concentration effect on the alleviation of cadmium stress.In this experiment,the effect of NO at 50μmol·L^-1 was the most significant.Further studies showed that 1000μmol·L^-1 of cadmium could reduce the relative water content and antioxidant enzyme activity of Kentucky bluegrass,increase the accumulation of MDA and proline,and inhibit the root growth.Applying 50μmol·L^-1 NO can effectively alleviate the damage of cadmium to seedlings,can increase relative water content,improve antioxidant enzyme activity,reduce the accumulation of MDA and free proline,increase total root length,total root surface area,number of root tips,number of root branches,and number of root crosses,and reduce the accumulation of cadmium in the overground and roots of plants.The results showed that cadmium treatment could aggravate the oxidative damage of Kentucky bluegrass and inhibit the growth of Kentucky bluegrass seedling,while adding exogenous NO could alleviate the growth inhibition of cadmium on Kentucky bluegrass by increasing antioxidant enzyme activity,reducing the accumulation of free proline and promoting root growth.4.Comparative transcriptome analysis was performed for the seedlings of Cd resistant cultivar（M）and Cd sensitive cultivar（R）under Cd stress.A total of 7022up-regulated and 1033 down-regulated transcripts were identified in M cultivar,whereas,only 850 up-regulated and 846 down-regulated transcripts were detected in R.Further transcriptional regulation analysis in M cultivar showed that Dof,MADS25,BBR-BPC,B3,bZIP23 and MYB30 might be the hub transcription factors in response to Cd stress due to the orchestrated multiple functional genes associated with carbohydrate,lipid and secondary metabolism,as well as signal transduction.Differential expressed genes involved in auxin,ethylene,brassinosteroid and ABA signalling formed signal transduction cascades,which interacted with hub transcription factors,thereby finally orchestrated the expression of multiple genes associated with cell wall and membrane stability,cell elongation and Cd tolerance,including IAAs,ARFs,SnRK2,PP2C,PIFs,BES1/BZR1,CCR,CAD,FATB,fabF and HACD.Additionally,post-transcriptional modification of CIPKs,MAPKs,WAXs,UBCs,and E3 ubiquitin ligases were identified and also involved in plant signalling pathways and abiotic resistance.The study could contribute to our understanding the transcriptional regulation and complex internal network associated with Cd tolerance in Kentucky bluegrass.5.To study the transcriptional regulation mechanism of exogenous NO on Cd tolerance of Kentucky bluegrass.The results showed that when the 4 treatments（CK、Cd、NO、Cd+NO）were compared,there were 22,730 differentially expressed genes,including 15,332 up-regulated genes and 7,398 down-regulated genes.The Kyoto Encyclopedia of Genes and Genomes（KEGGs）and Gene Ontology（GO）analyses revealed in the Cd+NO vs Cd comparison that the DEGs were primarily involved in the mitogen-activated protein kinase（MAPK）signaling,plant hormone signal transduction,phenylpropanoid biosynthesis,fatty acid metabolism,carbohydrate transport and metabolism,amino acid transport and metabolism,and biosynthesis-related pathways.In addition,the differentially expressed genes and metabolic pathways between Cd and Cd+NO treatment groups were analyzed to screen out key genes mediated by NO signal inresponse to cadmium stress.These genes include Glutamine-homocysteine methyltransferase,methionine-gamma-synthase,glutamine synthase,serine/threonine protein kinase,s-adenosine methionine synthase,peroxidase（POD）,UDP-glucose-6-dehydrogenase.These genes were also selected as candidate genes for NO to alleviate cadmium stress of Kentucky bluegrass.This study provides a basis for further exploration of Cd-related functional genes and regulatory genes of Kentucky bluegrass.