The Regulatory Mechanism Of Lead Accumulation In Radish (Raphanus Sativus L.) Taproot And Identification Of Related Genes

Radish（Raphanus sativus L.）is an important root vegetable crop belonging to Brassicaceae,which has high nutrition and medical value.Lead（Pb）is a nonessential metallic element posing high toxicity to living organisms.Melatonin（MT）is a tryptophan-derived natural product that plays a vital role in plant response to various abiotic stresses.Excessive Pb can harm human health through food chain.Therefore,how to effectively reduce the accumulation of Pb in vegetable crops through MT has become an important project.However,little information is available on MT-mediated regulatory network of Pb accumulation and detoxification in radish taproots.In this study,WGBS（whole-genome bisulfite sequencing）and strand special RNA-seq（ss RNA-seq）with the samples under Pb-free,Pb and Pb plus MT treatments were employed to construct a single-base resolution map of DNA methylation and long non-coding RNAs（lnc RNA）-involved regulatory network in radish plants.Based on radish genome database,identification of stress-responsive gene families were performed and their temporal and spatial expression profiles under abiotic stress were analyzed using radish RNA-seq data.Plant overexpression vector was constructed to validate the biological function of Rs HMA3 and Rs PCS2 genes in heavy metal stress response by transgenic technology.The main results obtained were as follows.1.A radish advanced inbred line‘NAU-YH’was used to study the effect of MT with different concentrations on the Pb accumulation,physiology and biochemistry in radish.The result showed that the Pb contents of both leaves and roots were significantly decreased after MT treatment.The Pb content under 50μmol/L MT treatment was 0.18 times and 0.40 times compared with Pb treatment in leaves and roots,respectively.In addition,the antioxidant enzyme activity of ascorbate peroxidase（APX）and glutathione reductase（GR）were increased under Pb stress with different melatonin concentrations,both of which reached the maximum level at50μmol/L MT.The lnc RNA library of radish under Pb-free treatment（Control）,Pb treatment（Pb200）,and Pb plus MT treatment（10、25、50、100 and 150μmol/L）were constructed.11,265 lnc RNAs were identified and classified into long intergenic non-coding RNAs（linc RNAs,8833）,antisense lnc RNAs（1298）and sense＿overlapping lnc RNAs（1134）.The MT treatment significantly changed the expression level of several genes,among which Pb-induced down-regulated genes were up-regulated after MT treatment,while the Pb-induced up-regulated genes was down-regulated after MT treatment.According to the targeted relationship of mi RNA-m RNA and mi RNA-lnc RNA,the regulatory network of lnc RNA-mi RNA-m RNA was constructed.It was noted that TCONS＿00177454 and Rs PDR12,targeted gene of mi R164c-5p,were up-regulated under Pb treatment and Pb plus 50μmol/L MT treatment.Furthermore,two proteins（Rs FBA8 and Rs HIPP26）interacted with Rs ASMT2 were found via yeast two-hybrid（Y2H）and bimolecular fluorescence complementation（Bi FC）.Expression analysis showed that Rs FBA8 and Rs HIPP26 were down-regulated under Pb treatment and up-regulated after MT treatment,implying that they may be involved in MT-mediated Pb detoxification and homeostasis in radish.2.The DNA methylation libraries of radish taproot under Pb-free,Pb and Pb plus 50μmol/L MT（Pb＿50MT）treatments were constructed,and WGBS analysis showed that the methylation level was increased under Pb stress,while an overall loss of DNA methylation was observed under 50μmol/L MT treatment.Among Control,Pb200 and Pb＿50MT,the proportion of m Cs in the CG context（～45.43%,45.10%and 44.14%）is higher than that in CHG（～26.10%,26.16%and 26.25%）and CHH（～28.47%,28.74%and 29.60%）.Heterochromatic regions with high density of transposable elements（TEs）were strongly methylated,while reduced methylation level was observed in gene-rich euchromatic regions in all three treatments.Differential analysis showed that 11,930,11,485 and 13,100 differentially methylated regions（DMRs）were obtained in Pb200 vs Control,Pb＿50MT vs Pb200 and Pb＿50MT vs Control,respectively.Among these,the methylation level of Pb200 vs Control hyper-DMRs was lower in Pb＿50MT than in Pb200.The DNA demethylase Rs DME and Rs ROS1genes were significantly up-regulated in Pb＿50MT,indicating that MT-mediated DNA demethylation may alter the expression of heavy metal（HM）-related genes and transcription factor（TF）with the participation of Rs ROS1 and Rs DME,which leads into low Pb accumulation and detoxification in radish.3.A total of 33 Rs HSF genes were identified from the radish genome,which were classified into group A（21）,B（10）and C（2）.Chromosomal localization analysis revealed that 28 Rs HSF genes were located on nine chromosomes.Quantitative real-time polymerase chain reaction（RT-q PCR）indicated that 9 Rs HSF genes were significantly differentially expressed under Pb stress,indicating that they may play a vital role in response to Pb stress of radish roots.Based on the whole genome data,28radish Rs ZIP genes were obtained,which were divided into 3 categories.Expression analysis showed that Rs ZIP12a was up-regulated under Cd（100mg/L and 200mg/L）and Pb（1000mg/L）stresses,while most Rs ZIP genes were up-regulated under200mg/L Cd Cl₂ and 1000mg/L Pb（NO₃）₂ treatment,such as Rs ZIP12b,Rs ZIP10b and Rs ZIP2.Moreover,13 Rs HMA genes were obtained from the radish genome.Under different heavy metal stresses,Rs HMA3 gene was up-regulated under Cd,Pb and Cr stresses,while Rs HMA4 gene was up-regulated under Pb stress.Furthermore,the Rs HMA3 and Rs PCS2 genes were overexpressed in tobacco,and the plant growth of T2 generation transgenic showed no significant change under 15mg/L Pb（NO₃）₂treatment.However,the root growth of wild-type plants was significantly inhibited under 30mg/L Pb（NO₃）_2,while the transgenic plants showed Pb tolerance,indicating that the Rs HMA3 and Rs PCS2 genes could enhance Pb tolerance of radish plants.These results could provide a fundamental foundation for investigation of regulation network and molecular mechanism underlying MT-mediated Pb stress response in radish.In conclusion,on the one hand,MT affects the transcript abundance of target genes（TCONS＿00177454 and Rs PDR12）by regulating the expression of mir164c-5p.In addition,Rs HIPP26 and Rs FBA8 interacted with Rs ASMT2 were up-regulated under exogenous MT treatment,which affected Pb accumulation and detoxification of radish taproot.On the other hand,MT-mediated DNA demethylation leads to low Pb accumulation and detoxification by changing the expression of HM related genes and TFs.