The Regulatory Role Of Responsive MiRNAs And Target Genes Of Wheat Roots Exposed To Phenanthrene In The Antioxidase System And Plant Growth

Polycyclic aromatic hydrocarbons(PAHs)are common organic pollutants in the environment.They are difficult to be removed because of high hydrophobicity and strong combination with soil particles.PAHs can enter the roots,stems,leaves,fruits or grains of plants along with absorption and transport of water and nutrients.They accumulate in human and animal bodies through the food chain,thus threating the health of humans and animals.MiRNAs(microRNAs)are a class of non-coding small RNA with a length of 18-30 nucleotides,which play an important regulatory role in plant growth and development and in response to environmental stress.In this paper,wheat is our research material and phenanthrene is the model of PAHs.We studied the differential expression of miRNAs and their target genes in wheat roots exposed to phenanthrene.Changes in wheat root morphology,physiological and biochemical activities,and transcription levels of gene expression were observed and measured.It is helpful to clarify the regulatory mechanism of miRNAs in wheat roots on response to phenanthrene exposure.The main research results are as follows:1.We compared the changes of miRNAs before and after phenanthene treatment(48 h)by high-throughput sequencing technology.Overall,the expression of miRNAs with the length of 18-30 nt changed a lot,in detail:(1)miRNA family in wheat roots obviously changed compared with the control group.The expression of many miRNAs families rose or lowered,and some members of the families increased or decreased.Non-conservative family had more changes then conservative family,because they were sensitive to environmental changes.(2)11 differentially expressed miRNAs in wheat roots were detected under phenanthrene treatment,including 6 conservative miRNAs(miR156,miR164,miR171a,miR398,miR531 and miR1121)and 5 nonconservative miRNAs(miR5048-5p,miR9653b,miR9678-3p,miR9773 and miR9778).In these miRNAs,Four(miR156,miR164,miR171a and miR9678-3p)were significantly up-regulated,and seven miRNAs(miR398,miR531,miR1121,mir50485p,miR9653b,miR9773 and miR9778)were down-regulated markedly.In addition,to ensure the accuracy of sequencing,the expression of these 11 miRNAs at 3 phenanthrene concentrations(0,0.5,and 1 mg L-1)and 4 time points(0,12,24,and 48 h)were further verified by miRNAs fluorescence quantitative PCR.(3)250 target genes of 11 miRNAs were detected with the help of psRobot and psRNATarget,including 152 target genes belonging to 6 conserved miRNAs and 98 target genes belonging to 5 nonconserved miRNAs.140 genes were screened out due to their possible regulation on plant growth and development cell component synthesis and transcription.We surmised that they may play an important role in response to phenanthrene exposure in wheat.(4)Common miRNAs with high expression levels and their target genes were identified.It is found that when wheat root was exposed to phenanthrene,miR156 and miR164 regulated the growth and development of wheat by targeting SPL(SQUAMOSA promoter-binding protein-like)and NAC(NAM/ATAF1/CUC),respectively.miR398,miR1121,and miR9773 affected the antioxidant system and metabolism by targeting CSD1/CSD2(Cu/Zn superoxide dismutase,Cu/Zn-SOD),CAT-1(Catalase 1),and CYP450(Cytochrome P450),respectively.2.miR398 regulated the antioxidase in wheat roots by targeting CSD1/CSD2 to resist the oxidative stress caused by phenanthrenel exposure.However,it is not clear how miRNAs worked in this process.We measured SOD activity and concentration of O2-and H2O2,and isolated SOD isozyme by PAGE.We also determined the expression of miR398 products at different stages of biosynthesis by means of stem ring reverse transcription and fluorescence quantitative PCR to obtain the specific effect of phenanthrene exposure on miR398.The study results were as follows:(1)Total SOD activity of wheat roots was significantly enhanced under phenanthrene treatment,in which the activity of Cu/Zn-SOD was significantly higher than that of the control.SOD isozyme electrophoresis showed that the bands of cytosol Cu/Zn-SOD changed significantly under phenanthrene exposure,and its regulatory gene CSD1 was also significantly up-regulated,indicating that cytosol Cu/Zn-SOD played a major role in removing ROS(reactive oxide species)caused by phenanthrene exposure.(2)When exposed to phenanthrene,the O2-level in wheat roots increased sharply.Then CAT activity increased to resolve the H2O2.This indicates that phenanthrene exposure first caused the accumulation of O2-and then the increase of SOD(mainly Cu/Zn-SOD)activity in wheat roots,and finally they alleviate the toxicity caused by phenanthrene.(3)Under phenanthrene exposure,the product of pri-miR398 decreased,the premiR398 increased,and the mature miR398 decreased.It indicated that phenanthrene exposure affected the transcription of miR398 regulatory gene-MIR398,hindered the transformation of pri-miR398 to pre-miR398 and then to miR398,resulting in the accumulation of pre-mir398 and the reduction of mature miR398,thus weakening the silencing effect on CSD1 and ultimately inducing the synthesis of SOD.(4)WRKY(The N terminal contains the WRKYGQK amino acid sequence)and/or transcription factors may play an important role in the whole regulation process.3.It is certified that miR164 can regulate root growth and lateral root generation of wheat under phenanthrene exposure by targeting NAC transcription factor.We compared wheat root morphology before and after phenanthrene exposure.Root activity,MDA(malonaldehyde)concentration and LOX(lipoxygenase)activity were measured,LOX isozymes were isolated by electrophoresis,and genes related to root development were detected.The main results are as follows:phenanthrene treatment accelerated the senescence/death of wheat roots,and stimulated the occurrence of new roots.However it is difficult to compensate for the loss caused by old root senescence/death,due to the slower growth of new roots under phenanthrene exposure.The accumulation of phenanthrene in wheat roots caused a lot of ROS,and the increase of LOX activity and MDA concentration,which showed that lipid peroxidation was the main cause of root damage.miR164 was up-related by phenanthrene,enhancing the silence to NAC1,weakening the association with auxin signal,and inhibiting the occurrence of lateral roots.Phenanthrene also affected the expression of CDK(cyclindependent kinase)and CDC2(cell division cycle),the key genes in the cell cycle of pericycle cells,thereby affecting the occurrence and growth of lateral roots.In addition,the genes,NAM and NAC23,may also be involved in the growth and development of wheat roots exposed to phenanthrene.All the above results confirmed that miRNAs in wheat roots are involved in response to phenanthrene exposure.miR398 and miR164 regulate the antioxidant enzyme activity and root growth and development of wheat roots exposed to phenanthrene by targeting genes CSD1 and NAC1,respectively.These results provide not only theoretical basis for understanding the molecular mechanism of crop response to PAHs accumulation,but also strategies for strengthening phytoremediation in PAHcontaminated soil or water.