Zhentian Lei

Mass Spectrometry Strategies in Metabolomics

MS has evolved as a critical component in metabolomics, which seeks to answer biological questions through large-scale qualitative and quantitative analyses of the metabolome. MS-based metabolomics techniques offer an excellent combination of sensitivity and selectivity, and they have become an indispensable platform in biology and metabolomics. In this minireview, various MS technologies used in metabolomics are briefly discussed, and future needs are suggested.

Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots

BackgroundWhite lupin (Lupinus albus L.) roots efficiently take up and accumulate (heavy) metals, adapt to phosphate deficiency by forming cluster roots, and secrete antimicrobial prenylated isoflavones during development. Genomic and proteomic approaches were applied to identify candidate genes and proteins involved in antimicrobial defense and (heavy) metal uptake and translocation.ResultsA cDNA library was constructed from roots of white lupin seedlings. Eight thousand clones were randomly sequenced and assembled into 2,455 unigenes, which were annotated based on homologous matches in the NCBInr protein database. A reference map of developing white lupin root proteins was established through 2-D gel electrophoresis and peptide mass fingerprinting. High quality peptide mass spectra were obtained for 170 proteins. Microsomal membrane proteins were separated by 1-D gel electrophoresis and identified by LC-MS/MS. A total of 74 proteins were putatively identified by the peptide mass fingerprinting and the LC-MS/MS methods. Genomic and proteomic analyses identified candidate genes and proteins encoding metal binding and/or transport proteins, transcription factors, ABC transporters and phenylpropanoid biosynthetic enzymes.ConclusionThe combined EST and protein datasets will facilitate the understanding of white lupins response to biotic and abiotic stresses and its utility for phytoremediation. The root ESTs provided 82 perfect simple sequence repeat (SSR) markers with potential utility in breeding white lupin for enhanced agronomic traits.

LAP5 and LAP6 Encode Anther-Specific Proteins with Similarity to Chalcone Synthase Essential for Pollen Exine Development in Arabidopsis

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

A Two-dimensional Electrophoresis Proteomic Reference Map and Systematic Identification of 1367 Proteins from a Cell Suspension Culture of the Model Legume Medicago truncatula

The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.

Root-Microbe Communication through Protein Secretion

Biotic interactions in the rhizosphere are biologically important, and although many of those interactions have been well studied, the role of secreted proteins in the cross-talk between microbes and roots has not been investigated. Here, protein secretion was studied during the communication between the roots of two plants (Medicago sativa and Arabidopsis thaliana) and the bacterial symbiont of one of these species (Sinorhizobium meliloti strain Rm1021) and an opportunistic bacterial pathogen of A. thaliana (Pseudomonas syringae pv. tomato DC3000) using a proteomic approach. It was found that protein exudation in the M. sativa-S. meliloti interaction caused an increase in the secretion of seven plant proteins, such as hydrolases, peptidases, and peroxidases among others in two or more time points compared with the plant control. In addition, four proteins, all of bacterial origin, were increased 1.5-fold more in this interaction compared with S. meliloti alone. However, these proteins were not induced when M. sativa was inoculated with P. syringae DC3000. The interaction between A. thaliana and P. syringae DC3000 highly induced the secretion of several plant proteins related to defense soon after initial contact with P. syringae, but these proteins were not secreted in the incompatible interaction with S. meliloti. The results of this study reveal a specific, protein level cross-talk between roots and microbes. These results suggest that secreted proteins may be a critical component in the process of signaling and recognition that occurs between compatible and incompatible interactions.

Soybean Metabolites Regulated in Root Hairs in Response to the Symbiotic Bacterium Bradyrhizobium japonicum

Nodulation of soybean (Glycine max) root hairs by the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum is a complex process coordinated by the mutual exchange of diffusible signal molecules. A metabolomic study was performed to identify small molecules produced in roots and root hairs during the rhizobial infection process. Metabolites extracted from roots and root hairs mock inoculated or inoculated with B. japonicum were analyzed by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry. These combined approaches identified 2,610 metabolites in root hairs. Of these, 166 were significantly regulated in response to B. japonicum inoculation, including various (iso)flavonoids, amino acids, fatty acids, carboxylic acids, and various carbohydrates. Trehalose was among the most strongly induced metabolites produced following inoculation. Subsequent metabolomic analyses of root hairs inoculated with a B. japonicum mutant defective in the trehalose synthase, trehalose 6-phosphate synthase, and maltooligosyltrehalose synthase genes showed that the trehalose detected in the inoculated root hairs was primarily of bacterial origin. Since trehalose is generally considered an osmoprotectant, these data suggest that B. japonicum likely experiences osmotic stress during the infection process, either on the root hair surface or within the infection thread.

Metabolite identification: are you sure? And how do your peers gauge your confidence?

Metabolomics is still faced with several significant challenges which currently limit its full scientific potential. The identification of metabolites is essential to convert analytical data into meaningful biological knowledge. However, identification confidence can vary widely because the process of identification is complex and dependent on the analytical platform and robustness of the methods applied, as well as the databases and resources used. Confident and unequivocal structure identification requires significant effort, which is multiplied dramatically in non-targeted metabolomics studies where 10–100s of metabolites can be deemed as biologically important and require identification. Mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR) or integrated MS–NMR strategies (Dunn et al. 2013; Kind and Fiehn 2010; van der Hooft et al. 2011) provide much information for the identification of metabolites (e.g. 1D/2D-NMR and MS/MS).

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

Antifungal Activity of Citrus Essential Oils

Citrus essential oils (CEOs) are a mixture of volatile compounds consisting mainly of monoterpene hydrocarbons and are widely used in the food and pharmaceutical industries because of their antifungal activities. To face the challenge of growing public awareness and concern about food and health safety, studies concerning natural biopreservatives have become the focus of multidisciplinary research efforts. In the past decades, a large amount of literature has been published on the antifungal activity of CEOs. This paper reviews the advances of research on CEOs and focuses on their in vitro and food antifungal activities, chemical compositions of CEOs, and the methods used in antifungal assessment. Furthermore, the antifungal bioactive components in CEOs and their potential mechanism of action are summarized. Finally, the applications of CEOs in the food industry are discussed in an attempt to provide new information for future utilization of CEOs in modern industries.

Influence of Host Chloroplast Proteins on Tobacco mosaic virus Accumulation and Intercellular Movement

Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.