As sessile organisms, plants are exposed to diverse abiotic and biotic stresses, and thus have developed complex signaling mechanisms that orchestrate multiple stress responses. Plant peptides have ...recently emerged as key signaling molecules of stress responses, not only to mechanical wounding and pathogen infection but also to nutrient imbalance, drought and high salinity. The currently identified stress-related signaling peptides in plants are derived from proteolytic processing of protein precursors. Here, we review these protein-derived peptides and the evidence for their functions in stress signaling. We recommend potential research directions that could clarify their roles in stress biology, and propose possible crosstalk with regard to the physiological outcome. The stress-centric perspective allows us to highlight the crucial roles of peptides in regulating the dynamics of stress physiology. Inspired by historic and recent findings, we review how peptides initiate complex molecular interactions to coordinate biotic and abiotic stress responses in plants.
Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct ...disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.
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•First deep proteogenomic landscape of non-smoking lung adenocarcinoma in East Asia•Identified age, sex-related endogenous, and environmental carcinogen mutagenic processes•Proteome-informed classification distinguished clinical features within early stages•Protein networks identified tumorigenesis hallmarks, biomarkers, and druggable targets
Deep proteogenomic landscape of early stage lung adenocarcinoma in a cohort of mostly non-smokers reveals unique drivers and biomarkers, as well as gender-associated mutagenesis.
MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein ...degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.
Abstract
Endogenous peptide hormones represent an essential class of biomolecules, which regulate cell–cell communications in diverse physiological processes of organisms. Mass spectrometry (MS) has ...been developed to be a powerful technology for identifying and quantifying peptides in a highly efficient manner. However, it is difficult to directly identify these peptide hormones due to their diverse characteristics, dynamic regulations, low abundance, and existence in a complicated biological matrix. Here, we summarize and discuss the roles of targeted and untargeted MS in discovering peptide hormones using bioassay‐guided purification, bioinformatics screening, or the peptidomics‐based approach. Although the peptidomics approach is expected to discover novel peptide hormones unbiasedly, only a limited number of successful cases have been reported. The critical challenges and corresponding measures for peptidomics from the steps of sample preparation, peptide extraction, and separation to the MS data acquisition and analysis are also discussed. We also identify emerging technologies and methods that can be integrated into the discovery platform toward the comprehensive study of endogenous peptide hormones.
Proteolytic activation of cytokines regulates immunity in diverse organisms. In animals, cysteine-dependent aspartate-specific proteases (caspases) play central roles in cytokine maturation. Although ...the proteolytic production of peptide cytokines is also essential for plant immunity, evidence for cysteine-dependent aspartate-specific proteases in regulating plant immunity is still limited. In this study, we found that the C-terminal proteolytic processing of a caspase-like substrate motif "CNYD" within Pathogenesis-related protein 1 (PR1) generates an immunomodulatory cytokine (CAPE9) in Arabidopsis. Salicylic acid enhances CNYD-targeted protease activity and the proteolytic release of CAPE9 from PR1 in Arabidopsis. This process involves a protease exhibiting caspase-like enzyme activity, identified as Xylem cysteine peptidase 1 (XCP1). XCP1 exhibits a calcium-modulated pH-activity profile and a comparable activity to human caspases. XCP1 is required to induce systemic immunity triggered by pathogen-associated molecular patterns. This work reveals XCP1 as a key protease for plant immunity, which produces the cytokine CAPE9 from the canonical salicylic acid signaling marker PR1 to activate systemic immunity.
Electrospray ionization-mass spectrometry (ESI-MS) is used to analyze metal species in a variety of samples. Here, we describe an application for identifying metal species by tandem mass spectrometry ...(ESI-MS/MS) with the release of free metals from the corresponding metal-ligand complexes. The MS/MS data were used to elucidate the possible fragmentation pathways of different metal-deoxymugineic acid (-DMA) and metal-nicotianamine (-NA) complexes and select the product ions with highest abundance that may be useful for quantitative multiple reaction monitoring. This method can be used for identifying different metal-ligand complexes, especially for metal species whose mass spectra peaks are clustered close together. Different metal-DMA/NA complexes were simultaneously identified under different physiological pH conditions with this method. We further demonstrated the application of the technique for different plant samples and with different MS instruments.
• The direct analysis of phytosiderophores (PSs) and their metal complexes in plants is critical to understanding the biological functions of different PSs. Here we report on a rapid and highly ...sensitive liquid chromatography‐electrospray ionization‐quadrupole‐time of flight‐mass spectrometry (LC‐ESI‐Q‐TOF‐MS) method for the direct and simultaneous determination of free PSs and their ferric complexes in plants. • In addition to previously reported PSs – deoxymugineic acid (DMA), mugineic acid (MA) and epihydroxymugineic acid (epi‐HMA) – two more PSs, avenic acid (AVA) and hydroxyavenic acid (HAVA), were identified by this method in roots of Hordeum vulgare cv Himalaya and in root exudates under iron (Fe) deficiency. • The two identified PSs could be responsible for Fe acquisition under Fe deficiency because of their relative abundance and ability to form ferric complexes in secreted root exudates. • This LC‐ESI‐Q‐TOF‐MS method greatly facilitates the identification of free PSs and PS–Fe complexes in one plant sample.
The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we ...identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA‐insensitive coi1‐16 and in ET‐insensitive ein2‐1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation‐PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR‐3 and PR‐4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96‐RNAi plants demonstrated wild‐type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96‐silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.
Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties ...in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.
A new G‐quadruplex (G‐4)‐directing alkylating agent BMVC‐C3M was designed and synthesized to integrate 3,6‐bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various ...telomeric G‐4 structures (hybrid‐2 type and antiparallel) and an oncogene promoter, c‐MYC (parallel), were constructed to react with BMVC‐C3M, yielding 35 % alkylation yield toward G‐4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI‐MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on G‐4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC‐C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c‐MYC), respectively, as monoalkylated adducts and formed A15–C3M–A21 (H26), G12–C3M–G4 (H24), and G2–C3M–G4/G17 (c‐MYC), respectively, as cross‐linked dialkylated adducts. Collectively, the stability and site‐selective cross‐linking capacity of BMVC‐C3M provides a credible tool for the structural and functional characterization of G‐4 DNAs in biological systems.
Bind and bond: BMVC‐C3M selectively alkylates various G‐4 structures. Analysis of the intact alkylation adducts by ESI‐MS revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Both monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on the binding surroundings of different G‐4 topologies (see figure).
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