iTRAQ Technology Research Articles

Comparative proteomic analysis of ovaries from Huoyan geese between pre-laying and laying periods using an iTRAQ-based approach.

To explore the key regulatory genes and their translated products involved in Huoyan goose egg laying, we first applied the iTRAQ technology integrated with LC-MS/MS analysis to identify differentially abundant proteins in the ovarian tissue of Huoyan geese between the pre-laying and laying periods. Then, Gene Ontology (GO) enrichment and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses of the proteins were conducted. As observed, 403 proteins, which included 255 up-regulated and 148 down-regulated proteins, were identified. Some differentially abundant proteins, such as apolipoprotein B (apoB), retinol-binding protein 4 (RBP4), vitellogenin-2 (VTG-2), apolipoprotein II (apoVLDL-II), and fatty-acid binding protein 4 (FABP4), were found to be involved in lipid transportation and metabolic process, follicle development and final egg production, ovarian steroidogenesis, and steroid hormone biosynthesis. Subsequently, the modification of the abundance of 3 proteins, adiponectin, FABP4, and RBP4, was verified using Western blotting. Our findings might provide comprehensive protein expression information that can facilitate the understanding of the reproductive biology and improve the egg-laying performance of Huoyan geese.

ITRAQ-based quantitative proteomic analysis of the earthworm Eisenia fetida response to Escherichia coli O157:H7

Soil environment contaminated by Escherichia coli O157:H7 which come from the waste of infected animals. Earthworms can live in the pathogens-polluted soil by their innate immunity. How the proteins of earthworms E. fetida will response to E. coli O157:H7-contaminated-soil still unclear? To identify the defense proteins under E. coli O157:H7 stress, we performed a proteomic analysis of earthworm under E. coli O157:H7 exposure through an iTRAQ technology. In total, we found 283 non-redundant proteins, including fibrinolytic protease 1, lombricine kinase, lysozyme, gelsolin, coelomic cytolytic factor-1, antimicrobial peptide lumbricin-l, lysenin, and et al. The proteins participate in metabolic processes, transcription, defense response to bacterium, translation, response to stress, and transport. The study will contribute to understand why earthworm can live in the pathogens-polluted environment.

Proteomic Analysis of Exosomes Released from Heatstressed Hepatocytes Reveals Promotion of Programmed Cell Death Pathways

Background: Liver injury is a common feature of heat stroke (HS). Exosomes, which are recognized as a novel cell-cell communication pathway that harbour functional cargo, may be involved in the pathogenesis underlying HS. However, little is known about the role of hepatocyte-derived exosomes in HS liver injury and their protein expression patterns under HS. Methods: In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were applied to explore changes in the proteomic profiles of HS-hepatocyte exosomes. Findings: In total, 53 and 21 proteins were identified as up- and downregulated in the HS-exosomes. Based on bioinformatics analysis using gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) pathway database, we found that regulation of programmed cell death was the most significantly altered pathway. Next, a candidate protein (TRAIL, TNF-related apoptosis-inducing ligand) that played a central role in the target pathway was chosen for validation. Western blotting clearly demonstrated enrichment of TRAIL in the HS-exosomes. We also confirmed that serum exosomal TRAIL could be used as a clinical marker for disease severity assessment and prognostic prediction of HS liver injury. Interpretation: Taken together, this study presented the first comprehensive analysis of the HS-hepatocyte exosomal proteome, confirming the induction of HS liver injury by exosomes and revealing novel potential pathways and proteins awaiting further investigation for clinical application. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant No. 81471839, No. 81671896 and No. 81873583). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal experiments were conducted in compliance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, 1985 revision) and were approved by the Animal Care and Use Committee of the General Hospital of Guangzhou Military Command.

Effects of let-7e on LPS-Stimulated THP-1 Cells Assessed by iTRAQ Proteomic Analysis.

Previous studies have demonstrated that let-7e is associated with inflammatory responses. To date, the roles and mechanisms of let-7e have not been completely revealed.Therefore, we aim to identify proteins associated with let-7e overexpression and explore their functions in the immune responses, including in cytokine production. High-throughput isobaric tag for relative and absolute quantitation (iTRAQ) technology is used to provide the first genome-wide study of THP-1 cells transfected with let-7e mimic followed by lipopolysaccharide (LPS) stimulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and KEGG pathway enrichment analyses are used to analyze a large number of differentially expressed proteins (DEPs) associated with let-7e overexpression or LPS stimulation. Quantitative reverse transcription PCR (qRT-PCR) and 50% tissue culture infective dose (TCID50) assays are processed to confirm the relationship of let-7e and dengue virus replication. iTRAQ results show that let-7e is associated with the expression of anti-viral proteins. What's more, calcineurin subunit B type 1, an anti-tumor factor, is upregulated by let-7e after LPS stimulation. KEGG analyses identify that some DEPS associated with let-7e overexpression are involved in the measles and influenza A pathways, and LPS-stimulated proteins in THP-1 cells are mainly enriched in transcriptional misregulation in cancer pathway and hippo signaling pathway (multiple species). The results of qRT-PCRand TCID50 show that let-7e promotes dengue virus replication, which is in agreement with the iTRAQ results. These results provide molecular insights into the regulatory mechanisms of let-7e in cytokine expression, virus replication, and anti-tumor function.

Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics.

Tuberculosis (TB) is a major comorbidity in HIV patients as well as a serious co-epidemic. Traditional detection methods are not effective or sensitive for the detection of Mycobacterium tuberculosis at the early stage. TB has become a major cause of lethal on HIV patients. We employed isobaric tags for relative and absolute quantitation (iTRAQ) technology to identify the different host responses in HIV-noTB and HIV-TB patients’ sera. Given the diversity of HIV subtypes, which results in a variety of host responses in different human populations, we focused on the Chinese patients. Of the 25 proteins identified, 7 were increased and 18 were decreased in HIV-TB co-infected patients. These proteins were found to be involved in host immune response processes. We identified a candidate protein, endoglin (ENG), which showed an 4.9 times increase by iTRAQ and 11.5 times increase by ELISA. ENG demonstrated the diagnostic efficacy and presented a novel molecular biomarker for TB in HIV-infected Chinese patients. This study provides new insight into the challenges in the diagnosis and effective management of patients with HIV-TB.

New insight into ovary abortion during ovary development of hazelnut through a combined proteomic and transcriptomic analysis

Abstract Hazel (Corylus spp.) is the most economically important species in the Betulaceae family. Frequent ovary abortion during the prefertilization stage causes the high drop ratio of pistillate flowers and smaller fruit numbers in a fruit cluster, resulting in yield loss. To better understand the mechanism of ovary abortion formation in hazel, we conducted transcriptomic and proteomic analyses and identified genes and proteins that were altered in response to ovary abortion. A total of 89,846 unigenes and 4343 proteins were identified using RNA-seq and iTRAQ technology, respectively. Among them, 1895 unigenes and 710 proteins were differentially expressed and accumulated in the developing and abortive ovary respectively. Furthermore, we employed the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, revealing pathways that are significantly enriched in both transcriptomic and proteomic assays. These include the phenylalanine metabolic pathway, biosynthesis of phenylpropanoid, biosynthesis of secondary metabolites, and ascorbate and aldarate metabolism. Transcriptome-proteome integrative analysis revealed 42 correlations. Among these, 32 correlations with same changing trend (that are involved in metabolism stress response, vascular strand development, water transport, and seed development) are likely associated with abortive ovary formation. Importantly, ethylene (ETH), jasmonic acid (JA), and salicylic acid (SA) signal transduction pathways may contribute to the regulation of the abortive ovary formation via the up-regulation of ethylene-responsive transcription factor 1 (ERF1/2), jasmonate ZIM domain-containing protein (JAZ), transcription factor MYC2 (MYC2), transcription factor (TGA), and pathogenesis-related protein 1(PR-1). Our work will facilitate the identification of ovary abortion related genes and proteins and provide insights into the molecular mechanism of fruit development.

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

PurposeThe aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication.Experimental designThree HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx‐Cm6, and HBx‐Cm16) were cultured. ITRAQ technology integrated with LC‐MS/MS analysis was applied to identify the proteome differences among these three cell lines.ResultsIn brief, a total of 70 different proteins were identified among HepG2‐HBx, HepG2‐HBx‐Cm6, and HepG2‐HBx‐Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2‐HBx and HepG2‐HBx‐Cm6 compared with HepG2‐HBx‐Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx‐minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild‐type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx‐minus HBV mutant genome were not restored to levels that were observed with wild‐type HBV by transient HBx expression.Conclusion and clinical RelevanceBased on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication.

ITRAQ-Based Proteomics Analysis of Triptolide On Human A549 Lung Adenocarcinoma Cells

Background/Aims: Triptolide (TP) is a diterpenoid triepoxide extracted from the traditional Chinese medical herb Tripterygium wilfordii that exerts prominent broad-spectrum anticancer activity to repress proliferation and induce cancer cell apoptosis through various molecular pathways. We previously observed that TP inhibits the progression of A549 cells and pancreatic cancer cells (PNCA-1) in vitro. However, the complex molecular mechanism underlying the anticancer activity of TP is not well understood. Methods: To explore the molecular mechanisms by which TP induces lung cancer cell apoptosis, we investigated changes in the protein profile of A549 cells treated with TP using a proteomics approach (iTRAQ [isobaric tags for relative and absolute quantitation] combined with NanoLC-MS/MS [nano liquid chromatography-mass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools OmicsBean and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and were verified using western blotting. Apoptosis and cell cycle effects were analyzed using flow cytometry. Results: TP induced apoptosis in A549 cells and blocked A549 cells at the G2/M phase. Using iTRAQ technology, we observed 312 differentially expressed proteins associated in networks and implicated in different KEGG pathways. Gene Ontology (GO) analysis showed the overviews of dysregulated proteins in the biological process (BP), cell component (CC), and molecular function (MF) categories. Moreover, some candidate proteins involved in PARP1/AIF and nuclear Akt signaling pathways or metastasis processes were validated by western blotting. Conclusion: TP exerted anti-tumor activity on non-small cell lung cancer (NSCLC) A549 lung adenocarcinoma cells by dysregulating tumor-related protein expression. Herein, we provide a preliminary study of TP-related cytotoxicity on A549 cells using proteomics tools. These findings may improve the current understanding of the anti-tumor effects of TP on lung cancer cells and may reveal candidate proteins as potential targets for the treatment of lung cancer.

CLIC1 Promotes the Progression of Gastric Cancer by Regulating the MAPK/AKT Pathways

Background/Aims: Chloride intracellular channel 1 (CLIC1), which is a member of the chloride channel protein family, is associated with various human tumors. Recent studies have shown that CLIC1 is involved in the occurrence and development of gastric cancer (GC). However, the exact mechanism remains unclear in GC. Methods: Effects of CLIC1 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated by analysing 54 patients with GC, as well as human gastric cell lines SGC-7901 and MGC-803, utilizing proteomics, RT-PCR, Western blotting, flow cytometry, Cell invasion and migration assays and xenograft tumor models. Results: Our study shows that CLIC1 knockdown by targeted-siRNA markedly inhibits GC cell invasion and migration and induces apoptosis in vitro. In total, 54 differentially expressed proteins were identified in GC cells SGC-7901 after CLIC1 silencing by isobaric tags for relative isotope labeled and absolute quantitation (iTRAQ) technology, including integrin α1 (ITGα1) and ITGα3. The expression levels of ITGα3, ITGαv, ITGβ1 and Bcl-2 mRNA and protein were decreased significantly in GC cells after CLIC1 knockdown; ITGα1 and Fas were upregulated, but the level of survivin was not significantly different. GC growth and metabolism were decreased in vivo after CLIC1 silencing, but apoptosis was markedly increased. Further study showed that the expression levels of ITGα3, ITGαv and ITGβ1, as well as AKT-phosphorylation, ERK-phosphorylation and p38-phosphorylation, were reduced in vivo after CLIC1 knockdown, while ITGα1 was upregulated. Conclusions: We speculate that CLIC1 may play an important role in the progression of GC, and its mechanism may be related to the regulation of integrin family proteins, which leads to the sequential regulation of the PI3K/AKT, MAPK/ERK and MAPK/p38 pathways.

ITRAQ-based comparative proteomic analysis provides insights into somatic embryogenesis in Gossypium hirsutum L.

Key messageiTRAQ based proteomic identified key proteins and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton.Somatic embryogenesis, which involves cell dedifferentiation and redifferentiation, has been used as a model system for understanding molecular events of plant embryo development in vitro. In this study, we performed comparative proteomics analysis using samples of non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryo (SE) using the isobaric tags for relative and absolute quantitation (iTRAQ) technology. In total, 5892 proteins were identified amongst the three samples. The majority of these proteins (93.4%) were found to have catalytic activity, binding activity, transporter activity or structural molecular activity. Of these proteins, 1024 and 858 were differentially expressed in NEC versus EC and EC versus SE, respectively. Compared to NEC, EC had 452 and 572 down- and up-regulated proteins, respectively, and compared to EC, SE had 647 and 221 down- and up-regulated proteins, respectively. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that genetic information transmission, plant hormone transduction, glycolysis, fatty acid biosynthesis and metabolism, galactose metabolism were the top pathways involved in somatic embryogenesis. Our proteomics results not only confirmed our previous transcriptomic results on the role of the polyamine metabolic pathways and stress responses in cotton somatic embryogenesis, but identified key proteins important for cotton somatic embryogenesis and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton.

ITRAQ analysis of a mouse acute myocardial infarction model reveals that vitamin D binding protein promotes cardiomyocyte apoptosis after hypoxia.

The proteome profile changes after acute myocardial infarction (AMI) and the roles played by important protein species remain poorly understood. Here, we constructed a mouse AMI model by ligating the left coronary artery of male C57B/6J mice to investigate the molecular changes after AMI on the protein level. Total proteins of the left ventricle were extracted and quantitatively analyzed by isobaric tags using relative and absolute quantitation (iTRAQ) technologies. The transcript and protein levels of important genes were further validated using quantitative polymerase chain reaction and western blot. An oxygen and glucose deprivation/reperfusion cell model was constructed using H9C2 cells to further validate the expression patterns and functions of important proteins after hypoxia. Seven hundred seventy-six proteins were identified as differentially abundant proteins after AMI, of which 406 were accumulated, and 370 were reduced. Gene ontology enrichment analysis showed that the most enriched molecular function category terms were binding, including calcium ion biding, GTP binding, actin binding and lipid binding. The expression levels of vitamin D binding protein (VDBP) and its related proteins were increased in both left ventricular tissue and H9C2 cells after ischemia-hypoxia. Overexpression of VDBP in H9C2 cells reduced vitamin D receptor and promoted the cell apoptosis rate after hypoxia. Our data provided new insights into proteome profile changes after AMI and indicated that VDBP could promote cardiomyocyte apoptosis after hypoxia.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients

IntroductionRecurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). MethodsTo gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. ResultsIn this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. DiscussionThis study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.

ITRAQ-based quantitative proteomic analysis and bioinformatics study of proteins in retinoblastoma.

The aim of the present study was to analyze proteins in the aqueous humor (AH) of patients' retinoblastoma (RB), and investigate their potential role in RB using the comparative proteomic technique of isobaric tags for relative and absolute quantitation (iTRAQ) coupled with offline two-dimensional liquid chromatography-tandem mass spectrometry. A total of 0.1 ml AH was collected from 10 children with RB (mean age, 3.8 years; range, 2-5 years) and patients with senile cataracts (mean age, 70.4 years; range, 65-79 years), which was used as the control. iTRAQ was used to analyze proteins in the AH of patients and controls. Proteins with a fold change of >1.20 or <0.83 were considered to be significantly differentially expressed (with corrected P<0.05). The identified proteins were subjected to subsequent gene ontology (GO) analysis using the DAVID database. A total of 83 proteins that were expressed differently between the controls and patients' AH samples were identified using iTRAQ analysis. Of these proteins, 44 were upregulated and 39 were downregulated. On the basis of biological processes in GO, the identified proteins were primarily involved in glycoprotein, amyloid acute-inflammatory and defensive responses. Among these proteins, pigment epithelium-derived factor serves a potential role in the treatment of RB, and stimulated by retinoic acid 6 may serve as a potential protein involved in RB development. To the best of our knowledge, the present study is the first to identify 83 proteins associated with RB using iTRAQ technology. The results of the present study will aid in furthering the understanding of RB and developing novel therapy targets in the future.

Combined transcriptomic and proteomic analysis constructs a new model for light-induced anthocyanin biosynthesis in eggplant (Solanum melongena L.).

Light is a key environmental factor affecting anthocyanin biosynthesis. Our previous study demonstrated that "Lanshan Hexian" is a light-sensitive eggplant cultivar, but its regulatory mechanism is unknown. Here, delphinidin-3-[4-(cis-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside and delphinidin-3-[4-(trans-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside were identified as the main anthocyanin components in Lanshan Hexian by ultra-performance liquid chromatography-tandem mass spectrometry. Three time points of anthocyanin accumulation, including the start point (0day), fastest point (5days), and highest point (12day), were investigated by using ribonucleic acid sequencing and iTRAQ technology. The corresponding correlation coefficients of differentially expressed genes, and differentially expressed proteins were 0.6936, 0.2332, and 0.6672. Anthocyanin biosynthesis was a significantly enriched pathway, and CHI, F3H, 3GT, 5GT, and HY5 were regulated at both transcriptional and translational levels. Moreover, some transcription factors and photoreceptors may participate in light-induced anthocyanin biosynthesis like the known transcription factors MYB113 and TT8. The transient expression assay indicated that SmMYB35, SmMYB44, and a SmMYB86 isoform might involve in the light-induced anthocyanin biosynthesis pathway. Finally, a regulatory model for light-induced anthocyanin biosynthesis in eggplant was constructed. Our work provides a new direction for the study of the molecular mechanisms of light-induced anthocyanin biosynthesis in eggplant.

Identification of plasma biomarkers for distinguishing bipolar depression from major depressive disorder by iTRAQ-coupled LC–MS/MS and bioinformatics analysis

It is important to differentiate between bipolar disorder (BD) and major depressive disorder (MDD) in the first depressive episode because of the potential treatment implications. Previous studies have mainly focused on the different clinical features or pathological biomarkers to distinguish these two diseases; however, a better understanding of the proteomics profiling of BD may help aid future therapeutic strategies. Here, we applied isobaric tags for relative and absolute quantification (iTRAQ) technology combined with liquid chromatography-tandem mass spectrometry (LC–MS/MS) to identify differentially expressed proteins between MDD and bipolar depression (BP). In total, 30 MDD, 30 BP and 30 healthy subjects were included. Proteins from depleted plasma samples were digested into peptides, individually labeled with iTRAQ reagents, combined and subjected to LC–MS/MS and further bioinformatics analyses. Our results showed that 9 proteins were significantly altered between MDD and BP. Briefly, B2RAN2, B4E1B2, APOA1, ENG, SBSN and QSOX2 were up-regulated, whereas ORM1, MRC2 and SLPI were down-regulated. Most identified proteins were related to the immune system. The bioinformatics analysis showed that B2RAN2 (highly similar to vanin-1) was involved in the significantly enriched KEGG pathways “pantothenate and CoA biosynthesis” (P=0.009). B2RAN2 and ENG may play important roles in depression. They may serve as candidate biomarkers for distinguishing MDD and BP. Further validation and investigation are required to illuminate the roles of B2RAN2 and ENG in MDD and BP. The current study provided a potential and novel biomarker panel that may, in turn, aid the diagnosis of BD.

Proteomic tools for studying RPE functions

SummaryHuman embryonic stem cell‐derived retinal pigment epithelial cells (hESC‐RPE) provide a promising cell source for studying retinal development, for disease modelling, and retinal cell replacement therapies. Several research groups, including ours, have demonstrated that hESC‐RPE structure, function, and physiology resembles that of native RPE regarding cellular fine structure and expression of many RPE signature genes and proteins. To characterize the hESC‐RPE proteome in larger scale, we have compared hESC‐RPE protein expression to primary human RPE using isobaric tags for relative quantitation (iTRAQ) technology. The hESC‐RPE proteome reflected that of native RPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No adverse signs such as increased stress, proliferation, or retinal degeneration‐related changes were seen in hESC‐RPE, while proteins involved in key RPE functions such as visual cycle and phagocytosis were detected. Proteomics provides valuable tools for validation of hESC‐RPE, studying disease mechanisms, and discovery of new biomarkers and therapeutic targets.

Comprehensive transcriptomics and proteomics analyses of pollinated and parthenocarpic litchi (Litchi chinensis Sonn.) fruits during early development

Litchi (Litchi chinensis Sonn.) is an important fruit that is widely cultivated in tropical and subtropical areas. In this study, we used RNA-Seq and iTRAQ technologies to compare the transcriptomes and proteomes of pollinated (polLFs) and parthenocarpic (parLFs) litchi fruits during early development (1 day, 2 days, 4 days and 6 days). We identified 4,864 DEGs in polLFs and 3,672 in parLFs, of which 2,835 were shared and 1,051 were specifically identified in parLFs. Compared to po1LFs, 768 DEGs were identified in parLFs. iTRAQ analysis identified 551 DEPs in polLFs and 1,021 in parLFs, of which 305 were shared and 526 were exclusively identified in parLFs. We found 1,127 DEPs in parLFs compared to polLFs at different stages. Further analysis revealed some DEGs/DEPs associated with abscisic acid, auxin, ethylene, gibberellin, heat shock protein (HSP), histone, ribosomal protein, transcription factor and zinc finger protein (ZFP). WGCNA identified a large set of co-expressed genes/proteins in polLFs and parLFs. In addition, a cross-comparison of transcriptomic and proteomic data identified 357 consistent DEGs/DEPs in polLFs and parLFs. This is the first time that protein/gene changes have been studied in polLFs and parLFs, and the findings improve our understanding of litchi parthenocarpy.

Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease

BackgroundThe normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease.ResultsIn this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis.ConclusionsBy integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Response mechanism of the docosahexaenoic acid producer Aurantiochytrium under cold stress

Aurantiochytrium is a commercial docosahexaenoic acid (DHA) producer, and its DHA content can be significantly increased under cold stress. Given this response to low temperature, we examined proteomics changes in Aurantiochytrium under cold stress. We detected approximately 700 protein spots using two-dimensional gels, whereas using iTRAQ technology, we detected 4650 types of proteins and successfully identified >53%. The results indicated that cold stress inhibits the cellular energy supply from glycolysis and the TCA cycle, to ensure a sufficient supply of NADPH and ribose for anabolism. In contrast, the pentose phosphate pathway was not affected. With respect to lipid synthesis, low temperature led to a significant down-regulation and up-regulation of fatty acid synthase and polyunsaturated fatty acid synthase, respectively, and restricted the protein synthesis of diacylglycerol O-acyltransferase and phospholipid: diacylglycerol acyltransferase. These results show the preferential biosynthesis of polyunsaturated fatty acids and phospholipids by Aurantiochytrium, which collectively serve to increase cell survival rates in cold environments.

Physiological and quantitative phosphoproteome analyses of drought stress-induced mechanisms in Malus baccata (L.) Borkh.

Limited information is available on how fruit crops respond to moderate drought stress. In the present study, we investigated how Malus baccata (L.) Borkh. a drought-tolerant genotype apple rootstock, responds to moderate drought stress. Our results for enzyme activity under moderate drought stress indicated that M. baccata produces osmosis-regulating substances. The phosphoproteins in the leaves were analyzed using iTRAQ technology. In total, 269 unique phosphopeptides, 304 phosphorylated sites, and 219 phosphoproteins were quantitatively analyzed in M. baccata. Furthermore, we identified 46 phosphoproteins in M. baccata whose phosphorylation levels significantly changed (PLSC). Among them, 22 PLSC phosphoproteins were found to be involved in metabolic processes that included carbon and nitrogen metabolism. This suggests that a systematic response pattern was generated in M. baccata and moderate drought stress resulted in a new homeostasis of carbon and nitrogen metabolism. The 14 differentially expressed mRNAs encoding phosphoproteins were analyzed by quantitative real-time PCR. Our study is the first to analyze the phosphoproteome of M. baccata and provides insights into the partial molecular regulatory mechanisms of M. baccata under moderate drought stress.