Dataset Information

Proteome-wide identification and functional analysis of ubiquitinated proteins in peach leaves.

ABSTRACT: Ubiquitination is a critical post-translational modification machinery that governs a wide range of cellular functions by regulating protein homeostasis. Identification of ubiquitinated proteins and lysine residues can help researchers better understand the physiological roles of ubiquitin modification in different biological systems. In this study, we report the first comprehensive analysis of the peach ubiquitome by liquid chromatography-tandem mass spectrometry-based diglycine remnant affinity proteomics. Our systematic profiling revealed a total of 544 ubiquitination sites on a total of 352 protein substrates. Protein annotation and functional analysis suggested that ubiquitination is involved in modulating a variety of essential cellular and physiological processes in peach, including but not limited to carbon metabolism, histone assembly, translation and vesicular trafficking. Our results could facilitate future studies on how ubiquitination regulates the agricultural traits of different peach cultivars and other crop species.

SUBMITTER: Song Y

PROVIDER: S-EPMC7015887 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Proteome-wide identification and functional analysis of ubiquitinated proteins in peach leaves.

Song Yanbo Y Shi Xiaojing X Zou Yanli Y Guo Juanru J Huo Nan N Chen Shuangjian S Zhao Chengping C Li Hong H Wu Guoliang G Peng Yong Y

Scientific reports 20200212 1

Ubiquitination is a critical post-translational modification machinery that governs a wide range of cellular functions by regulating protein homeostasis. Identification of ubiquitinated proteins and lysine residues can help researchers better understand the physiological roles of ubiquitin modification in different biological systems. In this study, we report the first comprehensive analysis of the peach ubiquitome by liquid chromatography-tandem mass spectrometry-based diglycine remnant affinit ...[more]

PMID: 32051488

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Project description:BackgroundAphid attack induces defense responses in plants activating several signaling cascades that led to the production of toxic, repellent or antinutritive compounds and the consequent reorganization of the plant primary metabolism. Pepper (Capsicum annuum L.) leaf proteomic response against Myzus persicae (Sulzer) has been investigated and analyzed by LC-MS/MS coupled with bioinformatics tools.ResultsInfestation with an initially low density (20 aphids/plant) of aphids restricted to a single leaf taking advantage of clip cages resulted in 6 differentially expressed proteins relative to control leaves (3 proteins at 2?days post-infestation and 3 proteins at 4?days post-infestation). Conversely, when plants were infested with a high density of infestation (200 aphids/plant) 140 proteins resulted differentially expressed relative to control leaves (97 proteins at 2?days post-infestation, 112 proteins at 4?days post-infestation and 105 proteins at 7?days post-infestation). The majority of proteins altered by aphid attack were involved in photosynthesis and photorespiration, oxidative stress, translation, protein folding and degradation and amino acid metabolism. Other proteins identified were involved in lipid, carbohydrate and hormone metabolism, transcription, transport, energy production and cell organization. However proteins directly involved in defense were scarce and were mostly downregulated in response to aphids.ConclusionsThe unexpectedly very low number of regulated proteins found in the experiment with a low aphid density suggests an active mitigation of plant defensive response by aphids or alternatively an aphid strategy to remain undetected by the plant. Under a high density of aphids, pepper leaf proteome however changed significantly revealing nearly all routes of plant primary metabolism being altered. Photosynthesis was so far the process with the highest number of proteins being regulated by the presence of aphids. In general, at short times of infestation (2?days) most of the altered proteins were upregulated. However, at longer times of infestation (7?days) the protein downregulation prevailed. Proteins involved in plant defense and in hormone signaling were scarce and mostly downregulated.

Project description:Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only approximately 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of approximately 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), approximately 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.

Project description:Understanding the defence mechanisms used by apple leaves against Alternaria alternate pathogen infection is important for breeding purposes. To investigate the ultrastructural differences between leaf tissues of susceptible and resistant seedlings, in vitro inoculation assays and transmission electron microscopy (TEM) analysis were conducted with two different inoculation assays. The results indicated that the resistant leaves may have certain antifungal activity against A. alternate that is lacking in susceptible leaves. To elucidate the two different host responses to A. alternate infection in apples, the proteomes of susceptible and resistant apple leaves that had or had not been infected with pathogen were characterised using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). MS identified 43 differentially expressed proteins in two different inoculation assays. The known proteins were categorised into 5 classes, among these proteins, some pathogenesis-related (PR) proteins, such as beta-1,3-glucanase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and mal d1, were identified in susceptible and resistant hosts and were associated with disease resistance of the apple host. In addition, the different levels of mal d1 in susceptible and resistant hosts may contribute to the outstanding anti-disease properties of resistant leaves against A. alternate. Taken together, the resistance mechanisms of the apple host against A. alternate may be a result of the PR proteins and other defence-related proteins. Given the complexity of the biology involved in the interaction between apple leaves and the A. alternate pathogen, further investigation will yield more valuable insights into the molecular mechanisms of suppression of the A. alternate pathogen. Overall, we outline several novel insights into the response of apple leaves to pathogen attacks. These findings increase our knowledge of pathogen resistance mechanisms, and the data will also promote further investigation into the regulation of the expression of these target proteins.

Dataset Information

Proteome-wide identification and functional analysis of ubiquitinated proteins in peach leaves.

Publications

Proteome-wide identification and functional analysis of ubiquitinated proteins in peach leaves.

Similar Datasets

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