Study On The Molecular Mechanisms Of High/Low Cadmium Accumulation Rapeseed Cultivars Respond To Cd Stress

In recent years,soil pollution in China is one of most wide and severe in the world,and soil cadmium?Cd?pollution in farmland is becoming more and more common.Soil Cd pollution not only seriously threatens the plant and development,but also brings potential harm to human health through the enrichment of food chain.Therefore,it is of great practical significance and application prospects to ensure the agricultural safety from Cd contaminated soils and to remediate Cd contaminated soils by using the heavy metal hyperaccumulator.Oilseed rape?Brassica napus L.?,as one of the most important oil crops in China,has a long history of cultivation and extensive planting area,which plays an important role in the development of our national economy.In the present study,a couple of B.napus genotypes with contrasting Cd root-to-shoot translocation and accumulation abilities,named high-Cd accumulator and low-Cd accumulator were chosen based on numerous seed resources of oilseed rape cultivars in China and their own genetic characteristics.Then the biomass and Cd content of roots and shoots of P78 and P72 under Cd stress,Cd transport capacity of shoot,accumulation ability of roots and shoots,photosynthetic system,oxidative damage,antioxidant content and antioxidant enzyme activity were studied and analyzed.Genomic resequencing analysis,transcriptomics and qRT-PCR analysis were used to screen for the key functional gene BnNramp that caused the difference between these two genotypes in response to Cd stress.Subsequently,through the functional complementation analysis of transgenic plants and yeast,the key role of the Cd transporter BnNramp gene in the transport of Cd from root to shoot in rapeseed was elucidated.The main conclusions are now briefly stated as follows:1.Screening and identification of high or low Cd accumulation cultivars from39 B.napus genotypesThe shoot biomass and Cd concentrations of 39 B.napus genotypes treated with2.5 and 5?M Cd for 10 days were analyzed by hydroponics.Exposed to 2.5?M Cd,the shoot biomass of the 39 B.napus genotypes were in the range of 0.047-1.247 g plant^-1 DW,and the shoot Cd concentration were 17.854-276.601 mg kg^-1 DW.Exposed to 5?M Cd,the shoot biomass of the 39 B.napus genotypes were in the range of 0.025-0.837 g plant^-11 DW,and the shoot Cd concentration were 36.323-359.284 mg kg^-1 DW.With cluster analysis of the biomass and Cd concentration of shoots,as the comprehensive screening indexes,we defined genotype P78 as a potential high Cd accumulator and P72 as a low Cd accumulator.we found that the shoots Cd concentration of P78 and P72 were 100.724 and 18.419 mg kg^-1 DW under 2.5?M Cd treatments,139.792 and 46.350 mg kg^-1 DW under 5?M Cd treatments,respectively.However the biomass of both genotypes were almost same,indicating that P78 and P72 differ greatly in their ability to respond to Cd toxicity.2.Further determination of high Cd accumulator and the low Cd accumulator and the physiological responses to Cd stressPrevious studies have shown that P78 and P72 were also high and low Cd accumulator under potting conditions.Therefore,we screened P78 and P72 as our test materials.Under Cd stress,the high Cd accumulator P78 and low Cd accumulator P72 were consistently toxic to Cd,mainly represented by that the root and shoot biomass,root surface area,root volume and root diameter of P78 and P72 were no difference under Cd stress.However,the Cd concentration in the roots and shoots of P78 were much higher than that in P72,especially in shoot.Under 2.5,5 and 10?M Cd stress,the Cd concentration in shoot of P78 were 4.5 times,2.9 times and 1.7 times more than that of P72,respectively.The Cd accumulation amount of P78 was also higher than that of P72.The Cd TF value in P78 was considerably higher than that in P72,especially under lower Cd levels.The BCF in P78 were also higher than that in P72.The difference of BCF _{Root/Solution} between P72 and P78 decreased with increasing Cd concentration,and there is no difference at 10?M Cd treatment,but the BCF _{Shoot/Solution} between P78 and P72 were significantly different under Cd,especially 2.5?M Cd treatment conditions.These results indicate that P78 had stronger capability to translocate Cd from root to shoot,and had higher Cd accumulation ability in shoot when comparing with P72.Determination of other metal elements indicates that the absorption of other metal elements is also affected by Cd stress,especially Fe,Zn,Mg and Cu.The addition of Cd inhibits the absorption of Mg and Zn by roots and promotes the uptake of Fe and Cu.Therefore,from the perspective of the ability to transport Cd from the root to the shoots,P78 is a potential high Cd accumulator and while P72 as a low Cd accumulator.The photosynthetic efficiency between P78 and P72 investigated to show that the photosynthetic performance decreased after Cd treatments,except the intercellular CO₂concentration?Ci?was weakly increase under Cd stress.The chlorophyll contents,variation of net CO₂ assimilation?Pn?and water use efficiency?WUE?in P78 were significantly affected by Cd treatment when comparing with P72.We investigated the antioxidant system between P78 and P72.There was no difference in MDA content between P78 and P72,and the contents of MDA were not affected by different Cd levels compared with normal condition.Therefore,the MDA contents shown that P72and P78 were not suffered from more serious lipid peroxidation under Cd stress.H₂O₂content was higher in P72 than that in P78,and increased with the increase of Cd concentration.The activities of CAT,POD,GR and APX in the two genotypes were all enhanced under Cd stress,P72 showed higher activities of these than P78,that means P72 is a more sensitive genotype than P78.P72 will produce more physiological responses to Cd stress when exposure to Cd.Interestingly,P78 shown a higher activities of SOD than that in P72,although they were all gradually decreased in two genotypes under Cd stress.This further demonstrates that P78 has a stronger ability to respond to Cd toxicity.3.Genomic resequencing data analysis and transcriptome difference analysis of the high Cd accumulator and the low Cd accumulator genotypes under Cd stressTo identify the genomic variations between the high Cd accumulator and the low Cd accumulator genotypes under Cd stress,we performed whole genomic re-sequencing?WGS?of P78 and P72.Analysis of genome-wide resequencing data for600 rapeseed varieties,we found 5290000 SNPs and 1310000 InDels in the B.napus genome.In detail,among these,4589407 SNPs were identified across the 19chromosomes of P78.4270658 SNPs were identified across the 19 chromosomes of P72.We obtained a series of related SNPs loci information by analyzing the differences in SNPs of reported Cd uptake and transport-related functional gene families.Such as,the SNP of BnNRAMP genes between P78 and P72 were detected.We found that BnNRAMP1;4,BnNRAMP1;5,BnNRAMP2;1,BnNRAMP2;3,BnNRAMP2;4 and BnNRAMP3;1 have 1 to 4 base mutations between P78 and P72.Further,we analyzed the effect of SNPs of BnNRAMPs on amino acid coding.The encoded amino acid sequence is identical in the SNPs present in the coding sequence region.To further elucidate the differential expressed genes,especially Cd transporter genes,involved in Cd accumulation between P78 and P72,we conducted transcriptome analysis through high-throughput digital gene expression?DGE?sequencing of the P78 and P72 roots and shoots with/without Cd treatment,respectively.After removing low-quality reads and those containing adapter and poly-N,more than 24 million clean reads remained for each sample,among which more than 90%were mapped to the B.napus genome.In total,19892 and 26987differentially expressed genes?DEGs?between the two lines?P78 Vs P72?were identified in the roots and shoots under Cd stress,respectively.Among all DEGs,9537genes were upregulated and 10355 genes were downregulated in roots,10616 genes were upregulated and 16371 genes were downregulated in shoots.And 10174 genes were common expressed.They are mainly involved in the absorption,transport and distribution of Cd,antioxidant systems and the absorption and transport of other metal elements,etc.A gene ontology?GO?enrichment analysis allowed us to identify the major functions categories of the DEGs between P78 and P72.The DEGs could be grouped into three categories:biological process?BP?,cellular component?CC?and molecular function?MF?.No matter the roots or the shoots,cellular process,metabolic process and single-organism process are the most enriched GO pathways in biological processes;cell part and membrane,while binding,catalytic activity and transporter activity were the three most enriched in the MF category.To further understand the molecular interactions among the DEGs,we performed KEGG analysis.For DEGs between P78 and P72 under Cd conditions,pathways of metabolic was most enriched in the roots,while plant hormone signal transduction was the most enriched in the shoots.In addition,the pathways of membrane transport was enriched in both roots and shoots.We found that under the Cd stress condition,the BnNramp gene family showed significant differences between P78 and P72,which is different from the expression levels of other medium and trace element transport protein families.Therefore,we propose that the BnNramp gene family may play an important role in the transport of Cd from root to shoot in P78 and P72.4.phylogenetic analysis and functional identification of NRAMP genes in B.napusBy sequence tree analysis and structural analysis of the BnNramp gene family,we analyzed the tissue specificity of 18 NRAMPs in Brassica napus and the expression level of 18 NRAMPs in two cultivars under Cd stress.Comparatively,both BnNramp2;1 and BnNramp4;2 were significantly highly expressed in shoot of B.napus and were upregulated obviously in shoot of P78,slightly in P72,under Cd stress condition.Therefore,BnNramp2;1 and BnNramp4;2 were screened as the candidate genes.Heterologous expression of BnNramp2;1 and BnNramp4;2 in yeast and Arabidopsis,respectively.Furthermore,BnNramp2;1 and BnNramp4;2 can successfully complement the function of tonoplast localized Cd transporter YCF1.And when BnNramp2;1 and BnNramp4;2 were transferred in Arabidopsis atnramp mutants,the transgenic plants showed better growth rate than mutants under higher Cd stress conditions.And the transgenic lines have significantly higher TF values than Arabidopsis mutants.This indicates that BnNramp2;1 and BnNramp4;2 are the major functional genes responsible for the difference of Cd between P78 and P72 from root to shoot.This study screened and identified the Cd accumulation high-efficiency genotype P78 and the inefficient genotype P72.Subsequently,phenotypic analysis,physiological index analysis,genomics and transcriptomics analysis,functional gene identification and functional analysis were used to clarify the main mechanism of Cd accumulation efficient and inefficient genotype in response to Cd transport from root to shoot.This work provides an important theoretical support for further elucidating the mechanism of heavy metal Cd accumulation in rapeseed,and provides an important way for phytoremediation technology of rapeseed heavy metal pollution in farmland soil.