Research Of Zinc Multiomics Analysis And Molecular Ecology Mechanism Of Sedum Alfredii Hance

Zinc(Zn)is an essential element for the plant,but too much Zn leads to toxicity of the plant.Sedum alfredii Hance is a Zn/cadmium(Cd)co-hyperaccumulator.Therefore,it is an ideal candidate to study the Zn hyperaccumulation mechanism in plant.Study on Zn hyperaccumulation mechanism of S.alfredii not only provide the theoretical guidance for biofortification,but also the theoretical basis for Zn nutrition.Till now,the studies on Zn hyperaccumulation mechanism by S.alfredii mainly focused on physiological processes and gene function analysis of transporters,however,no information on global analysis in response to Zn.Research on evolution key factors of S.alfredii also enhance the knowledge of Zn hyperaccumulation mechanism.Hence,in this research,the comparative transcriptome was used to study the genetic variation of hyperaccumulating ecotype(HE)and non-hyperaccumulation ecotype(NHE)of S.alfredii.Transcriptome,proteome,metabolome and ionome were used to study the genes expression,proteins biosynthesis,metabolites accumulation and ions content of roots and shoots of two ecotypes of S.alfredii responding to Zn.Furthermore,all the omics results were combined to comprehensively analyze the Zn hyperaccumulation of S.alfredii.The research on multiomics of S.alfredii can boarden the scope of knowledge of Zn hyperaccumulation mechanisms,and also provide the huge biological information to study the Zn hyperaccumualtion further.The detailed results were given step-by-step as below:1.Comparative transcriptome was studied using the Illumina 2500 platform to sequence the HE and NHE plant.More than 90 million clean reads were obtained.A total of 149,668/319,830 single nucleotide polymorphisms and 12,691/14,428 simple sequence repeats of HE/NHE were identified.The branch-site model was used to identify 18 evolutional key factors,which were RNA polymerasell Rpbl C-terminal repeat,28S ribosomal RNA gene,proteasome,cysteine proteinase inhibitor,calmodulin,calcipressin,nuclear transcription factor Y,WRKY transcription factor,apolipoprotein,histidine kinase,receptor-like protein kinase,F-actin capping protein and vacuolar-sorting receptor.The evolutional key factors were mainly involved in the transcription and translation processes,protein metabolism process,calcium(Ca2+)pathway,stress response process and signal transduction process.2.Digital gene expression profiling was studied using the Illumina 2500 platform to analyze the transcriptomic response of S.alfredii.A total of 317,761 transcripts,89,733 significantly differentially expressed transcripts were obtained.Topmost 100 upregulated and downregulated transcripts from each group comparison were extracted and united to do the annotation analysis.The results showed that 160 of these transcripts were classified in DNA modification,RNA recognization and protein synthesis,occupying 14.8%.In the transporter classification,ZIP genes family was expressed dramatically;with constitutively 1360-fold higher expression in roots and constitutively 719-fold higher expression in shoots comparison to HE with NHE plant.Hence,SaZIP4 was chosen to study the detailed gene fuction.The transcription levels of SaZIP4 were significantly and constitutively higher in HE than those in NHE plant,in accordance with the transcriptome results.A subcellular localization analysis indicated that SaZIP4 was localized to the plasma membrane.Expressing SaZIP4 in the yeast mutant ZHY3 can reverse the Zn uptake deficiency.Transgenic Arabidopsis thaliana mutant zip4-2 expressing SaZIP4h reversed the Zn/Cd uptake defect and wild type A.thaliana ectopically overexpressing SaZIP4h displayed increased Zn accumulation both in roots and shoots.Hence,SaZIP4 is an important Zn/Cd transporter.3.The isobaric tags for relative and absolute quantification(iTRAQ)was used to analyze the proteomic response of S.alfredii.Atotal of 736 proteins,and 269 significantly differentially expressed proteins were obtained.All the significantly differentially expressed proteins were annotated.The results showed that 15.1%significantly differentially expressed proteins were classified into the groups of DNA,RNA,protein basic transcript and translation process,that 18.8%of significantly differentially expressed proteins were participated in oxidation process,and that 7.7%of significantly differentially expressed proteins were involved in metabolic pathways.4.The GC-MS and LC-MS platforms were used to analyze the metabolomic response of S.alfredii.A total of 346 metabolites,256 significantly differentially accumulated metabolites were obtained.All the significantly differentially accumulated metabolites were mainly involved in TCA cycle,aminoacyl-tRNA biosythesis,amino acid metabolism,biosynthesis of unsaturated fatty acids and purine metabolism.5.The ICP-MS,ICP-OES and CNS analyzer platforms were used to analyze the ions content of S.alfredii.Combining and analyzing the trascriptome,proteome,metabolome and ionome results,the results showed that HE S.alfredii plants control a stable Zn uptake through reduction of transcription level in roots of the gene expression level of transporters in roots and shoots.In the meanwhile,HE S.alfredii plants were potential to synthesize more proteins related to photosynthesis,antioxidant activities and metabolic pathway through up-regulating the translation process in shoots.The mass of Zn in shoots was potential to bind with the above-mentioned proteins.To maintain the Zn homeostasis in HE S.alfredii,HE S.alfredii tightly control the rational Zn uptake,improve the ability of antioxidation,improve the photosynthesis efficiency and maintain the normal metabolic pathway.Besides,the carbon metabolism pathway is the most enriched pathway of all the significantly differentially expressed genes and proteins.Its genes,proteins and metabolites were controlled by Zn,and correlated with ions in plant to maintain Zn and ions homeostasis.