| Polycyclic aromatic hydrocarbons(PAHs)widely exist in soil and water,which are carcinogenic to humans and animals and have toxic effects on plant growth.In order to improve plant resistance,it is of great significance to study the mechanisms of plant response to PAH stress.However,most of the aforementioned studies focused on the response to PAHs in herbaceous plants.Furthermore,the plant resistance mechanism to PAHs remain unclear.Phenanthrene is a tricyclic PAH with high concentration in the environment,which has become a representative of the interaction mechanism between plants and PAHs.In this study,S.viminalis was selected as the experimental material,and a hydroponic experiment was conducted to explore the effects of phenanthrene stress on plant height and physiological characteristics.Afterwards,willows were treated with phenanthrene(1.0 mg·L-1)for 16 days to study the influence on mineral elements,photosynthetic characteristics,osmotic regulatory substances,reactive oxygen and antioxidant substances.Meanwhile,S.viminalis was planted in PAH-contaminated soil,and the effects of PAHs stress on root exudates were analyzed by collecting rhizosphere soil.Moreover,we used widely targeted metabolome sequencing and transcriptome to study the changes of metabolites and gene expression in the root of S.viminalis under phenanthrene stress.This study was proposed to provide new insights into the toxic mechanism of PAHs stress on plants and the plant resistance mechanism against PAHs stress,which would be helpful to the plant resistance breeding and research on enhancing capacity of plants to remedy PAHs.The results showed that:1.According to the concentration screening test,plant height was inhibited and physiology significantly changed when the concentration of phenanthrene was higher than 1.0 mg?L-1.Under 1.0 mg?L-1 phenanthrene treatment,the absorption of P,K,Ca and Fe was significantly restrained in mineral elements.The light reaction was affected as a result of significantly decreased photochemical quenching(q P).Gs,Ci and rubisco activity decreased,leading to the reduction of CO2 supply and assimilation.Consequently,the dark reaction was inhibited.The chloroplasts and mitochondria of leaves were damaged under phenanthrene stress,and photosynthesis decreased.Oxygen free radicals(O2·-)was the main reactive oxygen causing membrane lipid peroxidation in the leaf and root.SOD,CAT and POD were effective antioxidant enzymes for scavenging reactive oxygen species in the leaf,but did not play a consistent role in root.Reduced glutathione(GSH)was effective antioxidant for scavenging reactive oxygen in the leaf and root,and participated in the detoxification to phenanthrene through the catalysis of glutathione s-transferase(GST).Proline and soluble sugar were important regulatory substances to resist phenanthrene stress of S.viminalis.2.After PAHs treatment,many hizosphere metabolites changed significantly,which mainly included organic acids,carbohydrates,proline,verbasoside and sodium L-ascorbic acid 2-phosphate.Organic acids contained(Z)-octadec-9-enoic acid,succinic acid,decanoic acid,ricinoleic acid,pentadecanoic acid,tridecanoic acid and(Z)-hexadec-9-enoic acid.Carbohydrates contained turanose and trehalose.3.Ninety metabolites(75 upregulated and 15 downregulated)significantly changed after24 hours'phenanthrene exposure.Seventy-nine metabolites significantly changed(55upregulated and 24 downregulated after 36 hours'phenanthrene exposure.The rising metabolites consisted of amino acids,nucleotides and derivatives,carbohydrates,organic acids,flavonoids,alkaloids and lipids.At 24 h,those that were downregulated were mostly lipids.At 36 h,however,lipids,amino acids,organic acids and flavonoids were downregulated.There were thirteen amino acids,three carbohydrates,six organic acids,four flavonoids and one phenolamides related to resist phenanthrene.Among them,epigallocatechin,pinocembrin and apigenin played an important role in antioxidant and pathogen resistance.Chrysoeriol O-malonylhexoside possibly prevented the first step metabolism of phenanthrene in plants.N,N'-(p-coumaroyl)-cinnamoyl-caffeoyl-spermidine was an important substance to resist phenanthrene stress.The changes of some lipids(such as the decrease of MAG and the rising of HPODE)may affect the stability of cell membranes.4.After phenanthrene treatment for 24 hours,the number of upregulated genes was close to downregulated genes,thirty-five metabolic pathways had been significant changed.At 36 h,the downregulated genes increased while the upregulated genes decreased,twenty-four metabolic pathways had been significant changed.The transcriptome data showed that phenanthrene induced some metabolic pathways rising.The sulfur metabolism was upregulated owing to conversion of sulfate to sulfite and sulfides strengthening.As a result of the sulfide and serine transformation enhancing,the cysteine synthesis rised.GSH1,GR1,APX2 and several genes encoding GST rised in glutathione synthesis involved in detoxification and antioxidant to phenanthrene.Furthermore,sucrose synthesis,galactose metabolism,taurine and secondary taurine biosynthesis,and flavonoids biosynthesis were significantly upregulated.The key regulatory genes were obtained.Meanwhile,phenanthrene inhibited the synthesis of phenylpropanoid and affected the expression of aquaporin and iron transporter.In hormone synthesis,phenanthrene mainly blocked the zein biosynthesis and the transport of cytokinins.Nitrogen metabolism was influenced by hindering the transport of nitrate and nitrite.The photosynthetic carbon fixation was affected by inhibiting regeneration of Ru BP.5.The comprehensive analysis of transcriptome and metabolome data demonstrated that the toxic mechanisms of phenanthrene to S.viminalis mainly included:restraining the biosynthesis of phenylpropanoid and zein in roots,leading some lipids degradation,and blocking the expression of aquaporin,iron ion transporter and cytokinin transporter.Nitrogen metabolism and carbon metabolism decreased.The resistance mechanism of S.viminalis on phenanthrene mainly comprised:enhancing the biosynthesis of sucrose,maleosaccharide,inositol galactoside and some amino acids to provide plants with energy substances to resist phenanthrene stress,and strengthening the antioxidant and detoxification ability to phenanthrene by the upregulation of glutathione biosynthesis.Furthermore,the biosynthesis of aromatic glucosinolates,flavonoids,phenolamides and organic acids played an important role in improving antioxidant capacity,pathogen resistance and maintaining cell stability of plants. |
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