Study On Molecular Mechanism Of Mulberry In Response To Cadmium Stress

Heavy metal（HM）pollution has become one of the most urgent soil ecological problem in China because of its toxicity,non-degradation and bioaccumulation,which seriously threatens the human’s life and health and agricultural economic development.According to the report on the State of the Ecology and Environment in China 2019 and 2020,the main pollutant affecting the soil environmental quality of agricultural land was HMs,of which cadmium（Cd）was the primary pollutant.According to the report on the National General Survey of Soil Contamination in 2014,the exceed ratio of Cd was 7%,ranking first among heavy metals.The secondary standard value of Cd concentration in agricultural soils in China is 0.25-0.6 mg/kg.Some studies showed the areas with high Cd concentration（>1 mg/kg）are mainly distributed in some southern provinces,such as Yunnan,Guangxi and Hunan.Reducing HM concentration in cultivated land and making safe use of contaminated land are important tasks of the Action Plan on Prevention and Control of Soil Pollution released in 2016.Therefore,it is the key and difficult point of control of soil HM pollution to find and develop green,efficient and sustainable methods for control of the large-scale HM pollution.At present,the treatment methods of soil HM pollution mainly include chemical,physical and biological remediation technologies.Although the repair time of traditional methods is short,but these methods,including guest soil method,lime improvement method,chemical leaching method,heat treatment and electric repair method,have high investment cost,low efficiency,resulting in soil structure damage,easy to cause secondary pollution.Phytoremediation refers to the process by which green plants remove contaminants from polluted soil or water through processes such as phytostabilization and phytoextraction.This method is becoming a hot research and development area in the world due to its characteristics of in-situ remediation,safety,economy and no secondary pollution.Early studies mainly focused on herbaceous hyperaccumulators,but they were difficult to apply because of their annual growth,small biomass and low economic value,which caused it was difficult to be widely applied in the remediation of HM pollution.In recent years,perennial woody plants with large biomass have attracted the attention of scientists in the field of phytoremediation.Mulberry is a kind of perennial economic forest with well developed root systems,high biomass,widespread distribution,being readily adaptable and being capable of withstanding pruning.Mulberry leaves are the main feed of silkworm,and the main economic product silk is used for textile rather than food.The control of soil HM pollution by mulberry planting and sericulture system has aroused the interest of environmental pollution researcher and gradually become a hot spot in this field.When growing in Cd contaminated soil,most Cd in mulberry accumulates in the roots,and the Cd concentration in different parts of mulberry was root>stem>leaf;Most Cd in silkworm is excreted in the excrement,and the Cd concentration of different parts of silkworm was excrement>larva>cocoon.Based on literature review,data extraction and statistical analysis of published papers,the following conclusions were drawn:Perennial mulberry trees had a good tolerance to Cd,and the maximum tolerance to Cd in soil was 22.3～37.0 mg/kg.The maximum tolerance of silkworm to Cd in mulberry leaves varied greatly（0.49～6.48 mg/kg）.The weighted average Cd enrichment coefficients of roots,stems and leaves（Cd concentration of tissue/Cd concentration of soil）were 0.84,0.24 and 0.09,respectively.The weighted average Cd enrichment coefficients of excrements,larvae and cocoons（Cd concentration of parts/Cd concentration of mulberry leaves）were 1.51,1.23 and 0.14,respectively.The limit of Cd in the feed hygiene standards（GB 13078-2017）was 2.0 mg/kg,and the limit of Cd in the technical requirements of ecological textiles（GB/T 18885-2020）was 0.1 mg/kg.It was calculated that the limit of safe concentration of Cd in soil corresponding to safe utilization of mulberry for feed and cocoon production was 22.2 mg/kg and 7.94 mg/kg,respectively.Based on the calculation of the reported data,the number of samples in Cd contaminated soil（>0.25 mg/kg）less than 22.22 and 7.94mg/kg accounted for 98.39%and 91.57%of the total number of Cd contaminated samples,indicating that mulberry planting and sericulture are theoretically suitable for the safe utilization of more than 90%of Cd contaminated soil.In addition to mulberry planting and sericulture,in production practice,branches are cut every year,most of which are used as fuel or papermaking,etc.These processes make part of the Cd absorbed by mulberry trees removed from the soil,thus reducing the concentration of Cd in the soil.Therefore,in the relatively long process of remediation of heavy metal pollution in large areas of soil,safe utilization of Cd-polluted land through mulberry planting and sericulture can give consideration to ecological governance and economic development.All these results indicate that mulberry-silkworm system is a restorative system with great potential.Previous studies on the response of mulberry to Cd stress mainly focused on physiological analysis,and the understandings of related biochemical mechanisms are still limited.The lack of Cd stress response theory of mulberry severely restricts the exploitation of the potential of mulberry to effectively control soil Cd pollution.In addition,there are few reports on Cd stress tolerance and accumulation of different mulberry germplasm/variety resources,which seriously restrict the effectiveness of mulberry in the control of soil Cd pollution.Based on the above considerations,we have carried out our work from four aspects.i)Genome-wide identification,cloning and functional analysis of genes related to Cd uptake,transport,sequestration and chelation detoxification in mulberry were conducted.The key genes obtained can provide genetic resources for the creation of materials with different Cd accumulation characteristics.ii)Transcriptome data of reported plants under Cd stress were collected for meta-analysis to analyze the common key regulatory network and key genes of plant in Cd stress response,providing a basis for further elucidating the molecular mechanism of mulberry under Cd stress.iii)The evaluation of Cd tolerance and accumulation characteristics of different mulberry germplasm/variety was carried out,which provide theoretical and variety support for Cd contaminated soil control measures with different requirements.At the same time,cultivars with significant differences in Cd tolerance and accumulation were screened out to provide materials for the study of molecular mechanism of Cd stress response of mulberry.iv)Ionomics,transcriptomics and untargeted metabolomics analysis of mulberry cultivars with significant differences in Cd tolerance and accumulation characteristics were conducted to further clarify the molecular mechanism of mulberry in Cd stress response and dig out relevant key genes.The specific results are as follows:（1）Genome-wide identification,cloning and functional analysis of Cd transporter and phytochelatin synthase genes of mulberry1)Genome-wide identification and characterization of four gene families putatively involved in cadmium uptake,translocation and sequestration in mulberryIn this study,we identified 31 genes based on a genome-wide analysis of the Morus notabilis genome database.According to bioinformatics analysis,the four transporter gene families in Morus were distributed in each group of the phylogenetic tree,and the gene exon/intron structure and protein motif structure were similar among members of the same group.Subcellular localization software predicted that these transporters were mainly distributed in the plasma membrane and the vacuolar membrane,with members of the same group exhibiting similar subcellular locations.Most of the gene promoters contained abiotic stress-related cis-elements.The expression patterns of these genes in different organs were determined,and the patterns identified,allowing the categorization of these genes into four groups.Under low or high-Cd²⁺concentrations（30μM or 100μM,respectively）,the transcriptional regulation of the 31 genes in root,stem and leaf tissues of Morus alba seedlings differed with regard to tissue and time of peak expression.Heterologous expression of MaNRAMP1,MaHMA3,MaZIP4 and MaIRT1 in Saccharomyces cerevisiae increased the sensitivity of yeast to Cd,suggested that these transporters had Cd transport activity.Subcellular localization experiment showed that the four transporters were localized to the plasma membrane of yeast and tobacco.MaNRAMP1 and MaZIP4 were transient overexpressed in mulberry seedlings.Under Cd stress,the Cd concentration in roots and shoot of transgenic mulberry lines significantly increased compared with the control.2)Functional analysis of PCS genes involved in Cd chelation detoxification in mulberryUnder 30 or 100μM Cd²⁺stress,the relative expression of each of the two MaPCSs was induced in root,stem and leaf tissues within 24 h.Based on the analysis of total root length and fresh weight of seedlings,overexpression of Mn PCS1 and Mn PCS2 in Arabidopsis enhanced Cd²⁺tolerance in most transgenic individuals.The results of transgenic Arabidopsis lines overexpressing Mn PCS1and Mn PCS2 suggest that Mn PCS1 play a more important role in Cd detoxification than Mn PCS2.Cd²⁺concentrations in both shoots and roots of the transgenic Arabidopsis seedlings were higher than in wild type seedlings at two Cd²⁺concentrations.（2）Meta-analysis of reported Cadmium transcriptome in plantsTo extract generalization results from transcriptomic data across different plants and obtain some hub genes that respond to Cd stress,we carried out a meta-analysis of 32 published datasets.Cluster analysis revealed that plant species played a more decisive role than the media used and exposure time in the transcriptome patterns of plant roots response to Cd.The datasets from a Gramineae-like（GL）group were closer in clustering.838 DEGs were commonly Cd-regulated in at least nine of 18 GL datasets（frequency≥50%）.Gene ontology and KEGG pathway analyses revealed that oxidative stress-related terms and lignin synthesis-related terms were significantly enriched.Mapman analysis revealed that these common DEGs were mainly involved in regulation,cellular response,secondary metabolism,transport,cell wall and lipid metabolism.Comparative transcriptome analysis of six pairs of cultivars with high/low Cd accumulation traits showed that the related mechanisms of high/low Cd accumulation traits in different species might be different.（3）Evaluation of Cd tolerance and accumulation characteristics of different mulberry germplasm/varietyThe 10-day-old seedlings of the seven mulberry cultivars were treated with 20μM Cd Cl₂ for seven days.The Cd concentrations in shoots and roots of Fengchi（FC）were the highest,while those of Guisangyou12（G12）were the lowest.Moreover,G12 was more tolerant of Cd than FC.Considering the material requirements for omics studies,the 30-day-old FC and G12 seedlings were treated with or without 30 and 100μM Cd Cl₂ for seven days.The results showed that Cd concentration in FC roots was significantly higher than that in G12 roots at 100μM.（4）Ionomics,transcriptomics and untargeted metabolomics analysis of mulberry cultivars with significant differences in Cd tolerance and accumulation characteristicsG12 and FC were selected for comparative omics analysis.G12 was the cultivar with stronger Cd tolerance and lower Cd accumulation,while FC was the variety with weaker Cd tolerance and higher Cd accumulation.In ionomic analysis,there was no significant difference in ionomic characteristics between the two cultivars.Meanwhile,the distribution patterns of Na and Cd are most similar.In transcriptomics analysis,under the same conditions,the number of DEGs in G12was significantly lower than that in FC.Meanwhile,the number of DEGs was higher in roots treated with higher Cd concentration.Based on the results of GO and KEGG enrichment analyses,cell wall organization-related genes were significantly affected by Cd stress,while cell division-related genes were significantly down-regulated.Among the top 100 DEGs（based on Cohen’s D and fold change）between G12 and FC at the same Cd concentration,,we found multiple genes whose functions were associated with Cd response.In un-targeted metabolomics analysis,under the same conditions,the number of DMs in G12 was slightly higher than that in FC.Meanwhile,the number of DMs was higher in roots treated with higher Cd concentration.Compared with FC,the metabolites of G12 were less affected by Cd in phenylpropanoid,flavonoid and isoflavonoid biosynthesis pathways.Notably,the content of rutin in FC was significantly lower than that of G12.The above researches are helpful to provide theoretical support and candidate cultivars for mulberry application in Cd polluted soil,and candidate genes for mulberry genetic modification and molecular breeding in the future.