Molecular And Physiological Mechanisms Of Cadmium Absorption And Transportation In Salix Matsudana F. Umbraculifera

Increased anthropogenic activities,rapid industrialization,and modern agricultural practices have resulted in increased heavy metal cadmium(Cd)contamination in the environment during the last few decades.Cd is a non-essential element in plant growth and its compounds solubility,which has a strong ability to migrate in the soil-plant system.Phytoremediation is defined as the use of green plants to remove the heavy metal pollutants from the environment or render them harmless,which is low cost,safe and effective,in situ repair,sustainable process.At present,woody plants are the good candidate for the heavy metal contaminated soil repair materials for their high biomass,high growth rate,deep and highly branched root system and efficient uptake of heavy metals and translocation to the aerial parts.In this study,Salix matsudana(HL)as a control,its cultivarietas S.matsudana f.umbraculifera(MTL)was used as the research object to explore the variation in morphology,physiological,biochemical,and molecular response mechanism.After that,we employed the Illumina HiSeqTM2500 platforms to perform the comparative transcriptomic between the two species under Cd treatment at different time,and excavate the genes related to cadmium response,absorption,transport,detoxification.Finally,we investigated molecular regulatory mechanism.The main results were as follows:1.The homologies of ITS and matK between MTL and HL were 99.67%,and 99.63% respectively,in the molecular system evolution via sequence alignment.Under 100 ?mol L-1 CdCl2 treatment,there appeared to be some differences between MTL and HL in plant growth,root morphology,chlorophyll content,photosynthesis.2.Cd content of MTL is higher than that of HL in the same organization at the same time.With the extension of stress time,the difference was more significant.A strong net Cd2+ flux was observed after treatment for 24 h,and the variation trend of Cd2+ flux changes in MTL root apex was consistent with HL.The Cd2+ flux of MTL rhizosphere was higher than HL at the same root position,and accelerated gradually with the increase of stress time.Cd was allocated to root,stem,leaf,and vein of MTL in tissue level by histochemical assays.Furthermore,we found that Cd sequestration occurred mainly in vacuoles,cell wall,intercellular space by the trasnsmission electron microscopic observation and energy-dispersive X-ray microanalysis.Besides,there were some differences in functional groups,carrier or macromolecular substances that related to cadmium ion exchange,complexation,transport between the root,bark,wood,leaf of MTL and HL through fourier transform infrared spectroscopy..The tissues of MTL were promoted to coalesce,secret and transport organics(proteins,carbohydrates)to reduce the damage caused by Cd.In conclusion,MTL has stronger abilities on absorption,transportation and accumulation,and is a good candidate plant material for phytoremediation.3.Exposure to Cd elicited an oxidative burst in lipid peroxidation and ROS content in MTL and HL,and the oxidative stress level increased with the extension of stress time.Antioxidant enzymes activities and non-enzymes antioxidants contents showed a time-specificity and tissue-specificity under Cd treatment.According to the comparison of various physiological indicators,we found that MTL has stronger adaptability than HL under Cd stress.Plant hormones are well known for regulating many physiological processes and heavy metal stress tolerance.In this study,plant endogenous hormone auxin,abscisic acid,jasmonic acid and salicylic acid content variated greatly after the Cd stress.Jasmonic acid significantly increased,and it might play an important role in MTL response against Cd stress.4.To study the genes related to cadmium response,absorption,transport and detoxification,the roots of MTL and HL at different time under Cd stress were sequenced using the Illumina HiSeqTM2500 sequencing platform.After sequencing quality control received 152.46 Gb high quality Clean Data and 76 194 Unigenes were obtained,and the N50 was 1 427 bp.We found that responses to Cd stress were faster in HL than that in MTL according to the variation of DEGs expression at different time.However,the expressions of MTL DEGs were continuous and stable.DEGs of MTL and HL at different time stages were clustered in profiles based on gene expression pattern using STEM software.The profiles displayed a considerable difference in gene expression over time in response to Cd stress between MTL and HL.There were more genes involved in transport,detoxification biological processes in MTL roots.We chose 12 genes related to Cd stress responses,transport,detoxification,signal transduction,ATP-binding cassette transponer(ABC),bZIP transcription factor family(bZIP),Metallothionein(MT),Phytochelatin syntheses(PCS),Natural resistance-associated macrophage protein(Nramp)etc.to analyze their expression patterns.The results showed that the expression profiles of each gene in MTL and HL were different.This study will provide references to investigate their functions under Cd stress.5.To validate suitable reference genes for gene expression normalization in MTL under drought,salt,heavy metal stresses and six different tissues,we selected 11 candidate reference genes based on the transcriptome data.We identified ?-TUB2 and DnaJ as the most stable reference genes for normalization of gene expression under drought,salt,and heavy metal stress conditions according to the five systematic statistical algorithms analysis.Furthermore,the expression profiles of the target gene SmCAT validated that ?-TUB2 and DnaJ could be used as suitable reference genes.The stable reference genes identified in this study will facilitate accurate and consistent expression analysis of stress tolerance genes in willows under various abiotic stress conditions for functional genomic studies.