Lead Accumulation Characteristics And Its Regulatory Mechanism Of CAX/HMA Transporters Of Arabis Alpina In Lead-zinc Mining Areas

The threat of heavy metal pollution in soil to the safety of agriculture products and human health has be paid more and more attention.Meanwhile,the remediation of soil pollution is extremely urgent.Phytoremediation technology is a vital eco-friendly measure of repairing the soil polluted by heavy metals.The screening and application of native hyperaccumulators is the necessary basis of phytoremediation technology.It is of great theoretical and practical significance to study the process and mechanism of heavy metal uptake and accumulation of hyperaccumulators.Through laboratory simulation experiments and field investigation,the characteristics and mechanism of Pb uptake,accumulation and distribution of Arabis alpina were studied from levels of molecule,cell,tissue,individual,population and community.Based on the field investigation of the species diversity of A.alpina communities in different lead-zinc mining history areas,including the traditional smelter area(TST)with 40 years and the Sanduoduo village area(SDD)with 400 years,the adaptation and succession process of wild A.alpina communities in abandoned lead-zinc mining areas were studied.Through the simulation experiments,the effects of lead stress on seed germination,seedling growth,antioxidant enzyme activities,glutathione-ascorbic acid cycle,plant hormones and root exudates of A.alpina were investigated,so does the lead accumulation and subcellular distribution.Meanwhile,the transcriptome RNA-seq and real-time quantitative q RT-PCR were used to explore the molecular mechanism of response of A.alpina to lead stress.The results showed that:1.The contents of heavy metals in soil,the morphological and ecological characteristics of A.alpina were elucidated.In the two different investigation areas,the contents of heavy metals in the soil were Cd 9.92 mg/kg,Pb 660.41 mg/kg and Zn 4851.02 mg/kg in TST,while Cd 10.86mg/kg,Pb 535.95 mg/kg and Zn 4609.66 mg/kg in SDD,which showed no significant difference between TST and SDD.The contents of Cd,Pb and Zn in Arabis alpina exceeded the standards for hyperaccumulator,and its bio-accumulation factor and translocation factor were greater than 1.Therefore,A.alpina should be the hyperaccumulator of Cd,Pb and Zn.The root length,root volume and the ratio of root length to plant height were all positively associated with the contents of Pb in roots.There were significant differences in species diversity,morphological characteristics and heavy metal accumulation characteristics between A.alpina communities in two investigation areas,and temporal and spacial varieties were implied.The roots of A.alpina in TST had a tendency to grow underground and its roots had a tendency to become more thicker.The A.alpina in SDD had slenderer leaves and its plant height and branch number were all higher than those in TST.The main associated plants in the two areas were Sporobolus fertilis,Arenaria orbiculata and Eulalia speciosa.Shannon Wiener diversity index and Simpson diversity index were 0.56-2.04 and 0.44-0.95,respectively.Compared with A.alpina communities in SDD,the A.alpina communities in TST had more plant species,higher diversity index,lower ecological dominance and higher species evenness index.The Pb contents in shoots and roots of A.alpina in SDD was lower 84.50%and 80.05%than those in TST.In the long-term heavy metal stress environment,A.alpina has constantly evolved and gradually developed adaptability.2.It was found that the effects of Pb on the growth and development of A.alpina and the accumulation characteristics of Pb in cells and organelle.With the increase of Pb²⁺concentration(0,50,100,200,400 and 600 mg/L),the germination potential,germination rate and seed vigor index of A.alpina decreased gradually.Under 500 and 1000 mg/kg Pb²⁺treatments,the ratio of root biomass to shoot biomass increased from 0.34 to 0.48.The total root length,root diameter and root volume decreased by 43.7%,46.8%and 70.8%,respectively.The Pb contents in shoots and roots increased gradually.The bio-accumulation factor and translocation factor increased.Under the Pb²⁺stress,the total Pb contents and the Pb contents in subcellular part in roots and leaves increased gradually,while the Pb content in cell wall was the highest.With 500 and 1000mg/kg Pb²⁺treatments,the Pb contents of vacuoles soluble components in roots increased by200%and 340%,respectively,compared with the control.The Pb contents in leaf cell wall reached saturation and remained relatively stable.The Pb content in leaf organelles and vacuoles soluble components increased gradually.3.Studies on resistance physiology showed that the activities of antioxidant enzymes,the contents of endogenous hormones and low molecular organic acids were affected by lead stress.There was an dose-effect relationship between the antioxidant types and the dose of Pb stress.With the increase of Pb²⁺concentration(0,50,100,200,400 and 600 mg/L),the SOD,APX and CAT activities of A.alpina leaves increased gradually.With the 300 mg/kg Pb²⁺treatment,GSH contents and GR activities in leaves increased by 123.5%and 166.9%,while As A contents and APX activities increased by 145.5%and 33.3%,respectively,compared with the control.With 100and 300 mg/kg Pb²⁺treatments,the contents of plant hormones in leaves and roots increased.Compared with 100 mg/kg Pb²⁺treatment,the contents of gibberellin in roots increased by 15.3%.Considering the balance of endogenous hormones,the ratio of IAA/ABA in leaves and roots of A.alpina increased significantly,while the ratio of CTK/IAA greatly decreased under the Pb²⁺stress.With the increase of Pb²⁺concentration(0,500 and 1000 mg/kg),there was a decreasing trend in the contents of total organic acids,tartaric acid,citric acid,succinic acid,malic acid,acetic acid and oxalic acid in root exudates of A.alpina.Among the six low molecular weight organic acids,succinic acid accounted for the highest proportion(38-39%),followed by citric acid(28%-29%)and malic acid(25%).The resistance physiology of A.alpina to Pb stress was mainly manifested in the enhancement of antioxidant enzyme activities and the increase of glutathione and ascorbic acid contents.4.The changes of membrane transporter activities and the characteristics of lead absorption and transportation in A.alpina were revealed under lead stress.With 300 mg/kg Pb²⁺treatment,the activities of heavy metal ATPase(HMA)in leaves increased by 39.0%and51.9%,compared to the control and 100 mg/kg Pb²⁺treatment.The cation/proton exchanger(CAX)activities in leaves ad roots increased by 12.0%and 32.8%compared with the control.With100mg/kg Pb²⁺+25?mol/L Na₃VO₄or 25?mol/L DNP treatments groups,leaf HMA activities were significantly lower than those with 50?mol/L treatment groups.With the increase of Na₃VO₄concentration,root CAX activities decreased gradually.Under 100mg/kg Pb treatment,CAX activities of A.alpina leaves with 25?mol/L DNP or 50?mol/L DNP decreased by 16.3%and26.3%,respectively.With 100 mg/L Pb²⁺+25?mol/L Na₃VO₄or 25?mol/L DNP treatment groups,the total Pb contents in roots and leaves greatly decreased by 49.2%and 70.7%.With 100 mg/L Pb²⁺+50?mol/L Na₃VO or 50?mol/L DNP treatment groups,the total Pb contents in roots and leaves decreased significantly.Inhibition of Na₃VO₄or DNP decreased enzyme activities and the total Pb contents in roots and leaves,indicating that A.alpina could absorb and transport Pb actively.5.The molecular mechanism of lead transportation,accumulation and distribution was further revealed with analyzing the transcriptome of A.alpina.Through high-throughput sequencing RNA-seq technology,3.6GB of clean data was obtained.After the reads were assembled by Trinity software,a total of 121016 Unigene sequences were obtained.Among them,88622 sequences were annotated,accounting for 73.23%of all unigenes.The number of unigenes annotated to NR,Swiss-Prot,KOG and KEGG were 86261,74783,52400 and 63307,respectively.Compared with 100mg/L Pb treatment,the number of differential genes in leaves and roots of A.alpina from mining area was the most,which was 10649 in leaves(8051 up-regulated and 2598 down regulated)and 15592 in roots(13641 up-regulated and 1952 down regulated).With 100mg/L Pb treatment,the differentially expressed genes between leaf and root of A.alpina were the most,which was 12617(8229 up-regulated and 4398 down regulated).Go enrichment analysis of differentially expressed genes showed that the functions mainly focused on metabolic process,detoxification,supramolecular fiber,binding fixation,catalytic activity,electron carrier activity,transcription factor activity,protein binding,stimulation response and antioxidant activity.Compared with the control,the roots of A.alpina in mining areas and100mg/L Pb treatment showed gene expression up-regulation in cell detoxification,binding fixation,catalytic enzyme activity and electron carrier activity.Compared with the leaves,the differentially expressed genes in roots indicated the changes in genes related to binding fixation and extracellular matrix as well as the down-regulation of genes related to electron carrier activity.The differentially expressed genes related to lead resistance were mainly distributed in thiometabolism,flavonoid biosynthesis,plant hormone signal transduction,ascorbic acid metabolism and glutathione metabolism.The absorption and transport of Pb in A.alpina was an active process,which was regulated by two AaHMA2(Cadmium/zinc transporting ATPase HMA2 and P_1B-type ATPase 4-1)located in the cell membrane.AaCAX4 was the main transporter of CAX in leaves and roots of A.alpina,which was located on the vacuole membrane.Under Pb stress,the gene expression of AaCAX4,AaHMA2-1 and AaHMA2-2 in leaves and roots were significantly up-regulated.In conclusion,this study comprehensively analyzed the absorption,accumulation and distribution characteristics of Pb in A.alpina and discussed its physiological and molecular mechanisms,which could enrich the accumulation rules and resistance mechanism theories of hyperaccumulators to Pb.It is of great theoretical and practical significance to the phytoremediation of soil polluted by heavy metals.