- Differential expression pattern of the. - Results: This study examined the chemical forms of Cd in the roots of two wheat varieties (M1019 and Xinong20) by continuous extraction and analyzed differences in distribution characteristics of Cd in the root cell wall, cytoplasm, and organelles by elemental content determination and subcellular separation. - Furthermore, we conducted proteomics analysis of the roots of the two varieties under Cd pollution using mass spectrometry quantitative proteomics techniques. - In addition, the differentially expressed proteins in the two varieties were related to DNA replication and repair, protein metabolism, and the glutathione metabolism pathway.. - Conclusion: The results of this study improve our understanding of the mechanism of plant responses to Cd stress.. - In the natural environment, abiotic stresses including heat, cold, and heavy metals are not only harmful to the environ- ment, but also detrimental to plants and agriculture [2, 3].. - With the intensifica- tion of human activities such as mining and industrial ac- tivities and the excessive using of phosphate fertilizer, cadmium is released into the natural environment through geological and human activities. - Even at low concentra- tions, cadmium is one of the most toxic elements, which is due to high fluidity and bioavailability. - Therefore, cad- mium can more easily be absorbed in the underground part of plants and accumulated in the aerial part [5, 6].. - Wheat is one of the most important and the foremost food crops in China. - 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Roots rapidly respond to the pres- ence of Cd by forming a mechanical barrier [9, 10]. - The proportion of Cd in different chemical forms of M1019 variety was sodium chloride extraction state >. - 2), Cd in root tissues of M1019 and Xinong20 was mainly distributed in the cell wall, however the Cd content of M1019 was the lowest in organelles, and the Cd content of Xinong20 was lowest in cytoplasmic. - The original state- ment has been changed: The content of cadmium in the cell wall of M1019 was higher than that of xinong20, and the content of cadmium in the cell fluid and cytoplasm was lower than that of xinong20. - We conducted quality control testing of the obtained mass spectrometry data. - Second, most of the peptide lengths were distributed between 7 and 16 amino acid residues (Fig. - The results showed that the sample conformed to the rule of and the sample preparation reached criterion, so the sample can be used for next step of biological information analysis. - The closer Pearson’s coefficient is a measure of the linear correlation between sets of data. - 2 Distribution of cadmium in the cell wall, organelle components, and cytoplasmic After t-test, the significant difference at the level of 0.01. - 3 DAPs subcellular structure localization of M1019. - Therefore, we con- ducted statistical analysis of the distribution of proteins quantified by M1019 and Xinong20 in the GO secondary annotations. - The GO annotation is divided into three primary categories: the biological process, the cellular component, and the molecular function (Figs. - expression, the DEPs of M1019 were related to re- sponses to stress and carbon fixation. - 5 the mas error and peptide lengths of the sample. - Three biological replications were performed for proteomics analysis of the samples, and the left, center and right respectively represents the result of the first, second and third replications. - The dif- ferentially expressed proteins of Xinong20 were mainly re- lated to the balance of the ion, cell metabolism, and macromolecular biosynthesis. - Among the molecular process functions, the upregu- lated expression, the DEPs of M1019 were related to transferase activity, transferring glycosyl groups, and metal ion binding. - Among the cellular component process functions, the upregulated expression, the DEPs of M1019 were related to intrinsic and integral components of the membrane. - The DEPs of Xinong20 were related to the extracellular region. - For the downregulated expression, the DEPs of M1019 were. - However, the results showed that most of the DEPs in the MAPK signaling pathway of Xinong20 were related to pathogenic proteins. - A significantly greater en- richment of the glutathione metabolism pathway of M1019 than that in Xinong20 was observed (Fig. - For the upregulated expression, the results indicated that the DEPs of M1019 also enriched in protein processing in endoplasmic reticulum, alpha-linolenic acid metabolism, phenylalanine metabolism, galactose metabolism, and cysteine and methionine metabolism (Table 1).. - In addition, the DNA polymerase delta small subunit and replication protein 32 kDa sub- unit showed downregulated in the DNA replication, mis- match repair, and homologous recombination pathway (Table 2).. - Cd content distribution and chemical form distribution Most of the heavy metal absorption takes place through root cells, which distribute toxic ions to specific organs, tissues, or organelles. - Studies have shown that metal ions in Ni-rich plant cells, Zn hyperaccumulators and copper hyperaccumulators are mainly distributed on the cell wall, and the content in organelles is relatively small [17, 18]. - Ack 1 , Ack 2 and Ack 3 represent three biological replications of the control (nutrient solution only without cadmium).. - represent three biological replications of the control group (nutrient solution only without cadmium). - 7 Gene Ontology (GO) analysis of the upregulation of M1019 and Xinong20. - 8 Gene Ontology (GO) analysis of downregulation of M1019 and Xinong20. - which may be one of the factors of resistance to cad- mium in M1019. - In addition, the Cd content and per- centage of cell organelles of M1019 were lower than Xinong20, and the Cd content and percentage of cell wall were higher than Xinong20. - Therefore, M1019 can more effectively accumulate Cd in the cell wall and prevent excess Cd from entering cellular organelles. - According to the GO analysis, among the cellular component process, for the downregulated expression, the DEPs were detected in cytoplasmic part. - Some studies indicate that the ethanol extraction state and water-soluble Cd have the strongest migration activity, and the acid-. - In this study, the content of Cd in the ethanol-extracted and water-extracted state of M1019 variety was lower, and the content of acid- soluble Cd with weaker migration activity was higher than Xinong20. - However, the Cd with stronger migration activity is more likely to move upwards in Xinong20, thus caus- ing toxicity to the plants. - On the other hand, this may contribute to the accumulation of Cd in Xinong20 plants. - 9 Main DAPs enriched in the pathways. - Protein synthesis is substantially important for normal wheat development, new cell protein formation, protein degradation, and the output balance process. - Proteins are synthesized via transcription, translation, post- translational processing, and modification, which finally lead to the synthesis of the mature protein, which is strictly regulated in multiple steps [21]. - DNA recombinant repair protein can repair the damage caused by heavy metal pollution to the DNA molecules in the cells and re- stores its DNA structure and function. - DNA polymerase delta small subunit and replication protein A 32-kDa subunit showed downreg- ulated in the three pathways. - We found that components of the spliceosome pathway were downregulated in Xinong20 such as Table 2 The downregulation proteins in M1019 and Xinong20. - The hn-RNA is the initial prod- uct of DNA transcription, which requires further pro- cessing and modification of the spliceosome to form mRNAs with coding function. - Mitochondrial heat shock proteins play a role in the degradation of unstable and misfolded proteins and in the folding and refolding of proteins [27]. - We found that protein processing in the endoplasmic reticulum pathway was upregulated in M1019 such as heat shock cognate 70 kDa protein, 17.8 kDa class II heat shock protein, and cell division cycle protein 48 homo- log. - Heat shock proteins, which are in- volved in the translation process and in the post- processing process, were also downregulated in Xinong20. - Approximately 30% of the eukaryotic proteome is transmitted to the downstream compartments by endo- plasmic reticulum (ER) folding [29]. - This is critical to the vitality of all organ- isms [31, 32]. - In addition, the Sec61 complex is also the central component of the protein translocation apparatus of the ER membrane [33, 34].. - This ultimately leads to the sensitivity of Xinong20 to Cd toxicity.. - stress conditions, catalyzes the binding of glutathione to harmful substances, and transports the polymer to the vacuole through the action of ATP-binding transporter for detoxification [35]. - Decreased GS activity can promote the accumulation of Glu and contribute to the biosyn- thesis of the Cd-detoxifying substance, glutathione. - In this study, in the upregulation expression, the GST of M1019 and Xinong20 both showed an in- crease, but the M1019 showed a more increase. - In the downregulation expression, the GS of M1019 showed a downregulation expression, but GS of Xinong20 did not show the downregulation expression. - Therefore, glutathione and ABC may be one factor of the insensitivity of M1019 to Cd.. - By comparing the differences in Cd enrichment between two varieties, we show that the insensitive varieties M1019 can concentrate more Cd in the cell, and the mi- gration activity of Cd in the roots is relatively weak. - Our results indicate that insensitive wheat Xinong20 can more fully utilize glutathione for detoxification, and the sensitive variety exhibits weaker DNA replication, protein synthesis and degradation. - When the second leaves of the seed- lings emerged, CdCl 2 was added to the culture solution to a final concentration of 50 μM for 24 h. - Three biological replications were set up in this study and the results and data analysis are based on three bio- logical replications.. - Distribution of cd. - The homogenate was centrifuged for 15 min at 3000 rpm, and the resulting precipitate included the cell wall components. - The supernatant was then centrifuged for 30 min at 12,000 rpm, and the resulting precipitate included the organelle components, and the supernatant comprised the cytoplasmic component [37, 38]. - method, and the Cd content of each sample was deter- mined by Hitachi Z200.. - Approximately 20 mL of the extraction agent was then added to keep the sample saturated, and recycled extrac- tion after extracting it in a 30 °C incubator for 18 h.. - Then, an equal volume of the same extraction agent was added into the beaker of the sample, and recycled ex- traction for the second time after extract 2 h and repeat the extraction twice. - The extract of the chemical analysis of Cd was evaporated nearly 20 mL and then was determined using AAS ZEEnit700 after digesting with HNO 3 -HCIO 4 method.. - The peptide was reconstituted in 0.5 M TEAB and processed according to the protocol provided in the TMT kit. - The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in a Q Exactive™ Plus (Thermo) coupled online to the UPLC.. - The m/z scan range was 350 to 1800 for full scan, and intact pep- tides were detected in the Orbitrap at a resolution of 70, 000. - Peptides were then selected for MS/MS using NCE setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. - of the peptide segment was set to seven amino acid resi- dues. - The quantitative method was set to tmt-6plex, and the FDR for protein identification and PSM identification was set to 1%. - MY.J was major writing contributor to the manuscript, DZ. - H were contributors to acquire and organize data and make a preliminary interpretation to the data. - 2015Z39, 2016X58), and the National Key Research and Development Project for the 13th Five-year Plan (No. - The funding body was not involved in the design of the study, analysis or inter- pretation of data or writing the manuscript.. - The datasets generated and analysed during the current study are available in the [ProteomeXchange] repository, [ProteomeXchange accession: PXD018179 and FTP Download: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/03/. - A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti . - The Dynamic Changes of the Plasma Membrane Proteins and the Protective Roles of Nitric Oxide in Rice Subjected to Heavy Metal Cadmium Stress.. - Cellular Compartmentation of zinc in leaves of the Hyperaccumulator Thlaspi caerulescens1. - Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex. - Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant. - The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe. - The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation
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