Molecular Mechanisms Of Lead Tolerance And Accumulation In Tartary Buckwheat(Fagopyrum Tataricum)

Lead?Pb?is one of the most toxic heavy metal persistent pollutants.Lead pollution has become a serious environmental problem in the world.Pb contaminated soil will seriously affect the germination,growth and development of plant?crop?seeds,and will seriously damage the human nervous,immune and reproductive systems after enter the food chain,and also has a cumulative nature.Phytoremediation of heavy metals in soil,including lead contamination,is considered to be the cheapest and most promising technique for remediation of heavy metal contaminated soil.The existing recognized heavy metal enrichment plants are generally characterized by small biomass and long growth cycle,so it is of great practical and theoretical value to screen and create Pb pollution-tolerant enrichment plants and to analyze their intrinsic enrichment and tolerance mechanisms.It is found that Tartary buckwheat is a high lead tolerance plant in the early screening and evaluation of lead tolerance of various plants.Tartary buckwheat has the characteristics of strong regional adaptability,short growth period and abundant biomass.At present,there has been no systematic study on Pb tolerance and enrichment of Tartary buckwheat,and its metabolic pathways and gene expression regulation mechanisms in response to lead stress have yet to be revealed.However,none of the genes and metabolic pathways involved in regulating the Pb stress response in tartary buckwheat have been identified.In this study,the Fagopyrum tataricum‘Jiujiang'cultivar was used as the material,and Pb was used to compare the localization of tartary buckwheat in different tissues and the determination of heavy metal content.On the basis of using Illumina sequencing technology the high quality of the buckwheat leaves the transcriptome analysis of lead stress data,identification of key genes associated with lead tolerance mechanism and approaches,and use of defect in yeast expression of heavy metal tolerance and expression of gene in Arabidopsis mutants validation of gene function,aims to provide theoretical basis for phytoremediation.The main results include:1. Lead exposure up to 10,000 mg/kg reduced height but had no other effect on the buckwheat plants.this was the highest concentration assayed in subsequent experiments.Furthermore,lead exposure caused no significant change in MDA content,GSH content,soluble protein and SOD activity indicating the treatments caused less damage to the leaf physiology under the lead treatment concentration range of 0-10000 mg/kg,it indicates that Tartary buckwheat has a strong tolerance to lead.In the present study,Transmission electron microscopy analysis of different tissues under lead stress showed that lead was distributed in different tissues of Tartary buckwheat,and lead ions were mainly distributed in vacuoles and inside cell walls of Tartary buckwheat leaves.The determination of lead accumulation in different tissue parts of tartary buckwheat by atomic absorption spectrometry showed that with the increase of lead concentration,the accumulative amount of lead in Tartary buckwheat leaves also increased significantly,with the maximum accumulative amount of 499.126±64.791 mg/kg,and the lead ion translocation factor?content of overground part/content of underground part?was about 0.8,which was close to the super-enriched plants.Therefore,Tartary buckwheat can be used as a potential phytoremediation of Pb-polluted soil.2. RNA deep sequencing?RNA-Seq?of tartary buckwheat leaves was performed on twoPb-treated samples,including 0 mg/kg?CK?,2000 mg/kg?Pb1?and 10,000 mg/kg Pb?Pb2?.In total,2400 up-regulated and 3413 down-regulated differentially expressed genes?DEGs?were identified between CK and Pb1,and 2948 up-regulated DEGs and 3834downregulated DEGs were generated between CK and Pb2,respectively.To identify unigenes that were induced by Pb stress,the three libraries were divided into two groups?CK vs Pb1 and CK vs Pb2?,and 4525 common DEGs were identified in these comparisons.In total,we identified 1641 upregulated and 2884 downregulated unigenes under Pb stress.GO enrichment analysis identified unigenes in tartary buckwheat under Pb stress were primarily associated with catalytic activity,binding,metabolic processes and membrane components.KEGG enrichment indicate that Pb stress in F.tataricum primarily influences pathways associated with energy metabolism,lipid metabolism,secondary metabolites,nonenzymatic antioxidants and oxidative phosphorylation.3. In this study,we found that the gene expression of the tandem CCCH Zinc finger protein14?Ft TZF14?was significantly upregulated in F.tataricum leaves under the Pb stress.We confirmed that the overexpression of Ft TZF14 could seriously inhibit the growth of the Pb sensitive yeast mutants?ycf1 and Arabidopsis.And the transient transformation of Ft ZFP14showed that it could rapidly promote the degradation of Rubisco in Tartary buckwheat leaves.Meanwhile,the ubiquitination inhibitor MG132 could significantly inhibit this degradation process.Furthermore,we have screened and identified an ubiquitin ligase enzyme Ft ATL42could interact with Ft TZF14.All the results indicate Ft ZFP14 was involved in the signal transduction process of the ubiquitin/26S proteasome mediated degradation of Rubisco.The results suggested that the growth inhibition of Tartary buckwheat under lead stress might be related to the ubiquitination degradation of Rubisco mediated by Ft ZFP14.4. On the basis of real-time quantitative verification of 3 differentially expressed genes?DEGs??Metal tolerance protein C2?Ft MTPC2?,Natural resistance-associated macrophage protein 3?Ft Nramp3?and Vacuolar cation/proton exchanger 1a?Ft VCE1a??upregulated under lead stress,we verified the gene functional complementarity by using the Pb sensitive yeast mutants?ycf1.The results suggested that the expression of Ft MTPC2,Ft Nramp3 and Ft VCE1a strongly increased the Pb tolerance in the Pb-sensitive yeast strain?ycf1.Further study indicates that Ft MTPC2,Ft Nramp3 and Ft VCE1a could enhance the tolerance of Arabidopsis thaliana to Pb.The results suggested that Ft MTPC2,Ft Nramp3 and Ft VCE1a are important in the process of Pb transport.We have screened and identified the interaction proteins of the heavy metal transporters?Ft MTPC2,Ft Nramp3 and Ft VCE1a?through the Yeast two-hybrid library of F.tataricum leaves under lead stress,then we have confirmed the interaction through Yeast two-hybrid?Y2H?and Bimolecular Fluorescent Complimentary?Bi FC?assays.Furthermore,we have analyzed the gene expression levels of the interaction proteins,which reflected the gene expression levels of IAA-alanine resistant 1?Ft IAR1?,Cadmium/zinc-transporting ATPase-HMA1?Ft HMA1?and Glutathione-dependent formaldehyde dehydrogenase?Ft FDH?proteins interacting with Ft MTPC2 were all up-regulated,and that indicates these proteins have the co-transport function to Ft MTPC2.For the protein interacting with Ft Nramp3,the gene expression level of Ft HMA3 increased significantly with the same expression pattern to Ft Nramp3;while the gene expression levels of Heat shock protein 70?Ft HSP70?and Zinc transporter 1?Ft ZRT1?decreased,which may be antagonism with Ft Nramp3 under lead stress.In addition,the gene expression of the protein(Mg²⁺/H⁺exchanger?Ft MHX?,Monothiol glutaredoxin-S7?Ft GRXS7?and Natural resistance-associated macrophage protein 6?Ft Nramp6?)interaction with Ft VCE1a did not significantly change,it is speculated that the gene expression are not affected by Pb stress.In summary,this study effectively proved that Tartary buckwheat has a high tolerance to Pb.Tartary buckwheat can be used as a soil remediation plant.We analyzed the tolerance and accumulation mechanism of Tartary buckwheat leaves to lead by transcriptome sequencing;meanwhile,we explored the important functional genes by expressing in yeast and Arabidopsis thaliana system.These results provide theoretical and research basis for Tartary buckwheat as a potential plant for phytoremediation of Pb-polluted soil.