TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

Genome mining reveals the biosynthetic potential of the marine-derived strain Streptomyces marokkonensis M10.

Marine streptomycetes are rich sources of natural products with novel structures and interesting biological activities, and genome mining of marine streptomycetes facilitates rapid discovery of their useful products. In this study, a marine-derived Streptomyces sp. M10 was revealed to share a 99.02% 16S rDNA sequence identity with that of Streptomyces marokkonensis Ap1T, and was thus named S. marokkonensis M10. To further evaluate its biosynthetic potential, the 7,207,169 bps of S. marokkonensis M10 genome was sequenced. Genomic sequence analysis for potential secondary metabolite-associated gene clusters led to the identification of at least three polyketide synthases (PKSs), six non-ribosomal peptide synthases (NRPSs), one hybrid NRPS-PKS, two lantibiotic and five terpene biosynthetic gene clusters. One type I PKS gene cluster was revealed to share high nucleotide similarity with the candicidin/FR008 gene cluster, indicating the capacity of this microorganism to produce polyene macrolides. This assumption was further verified by isolation of two polyene family compounds PF1 and PF2, which have the characteristic UV adsorption at 269, 278, 290 nm (PF1) and 363, 386 and 408 nm (PF2), respectively. S. marokkonensis M10 is therefore a new source of polyene metabolites. Further studies on S. marokkonensis M10 will provide more insights into natural product biosynthesis potential of related streptomycetes. This is also the first report to describe the genome sequence of S. marokkonensis-related strain.

Vitroprocines, new antibiotics against Acinetobacter baumannii, discovered from marine Vibrio sp. QWI-06 using mass-spectrometry-based metabolomics approach.

A robust and convenient research strategy integrating state-of-the-art analytical techniques is needed to efficiently discover novel compounds from marine microbial resources. In this study, we identified a series of amino-polyketide derivatives, vitroprocines A-J, from the marine bacterium Vibrio sp. QWI-06 by an integrated approach using imaging mass spectroscopy and molecular networking, as well as conventional bioactivity-guided fractionation and isolation. The structure-activity relationship of vitroprocines against Acinetobacter baumannii is proposed. In addition, feeding experiments with (13)C-labeled precursors indicated that a pyridoxal 5'-phosphate-dependent mechanism is involved in the biosynthesis of vitroprocines. Elucidation of amino-polyketide derivatives from a species of marine bacteria for the first time demonstrates the potential of this integrated metabolomics approach to uncover marine bacterial biodiversity.

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Analysis of the biosynthesis of antibacterial cyclic dipeptides in Nocardiopsis alba.

Nocardiopsis alba is frequently isolated from environment and has recently been suggested as a casual symbiotic actinobacterium of diverse invertebrates. Using activity-guided fractionation, we purified two antibacterial cyclic dipeptides, cyclo(ΔPhe-ΔLeu) (albonoursin) and cyclo(ΔmTyr-ΔLeu), from a culture of Nocardiopsis alba ATCC BAA-2165. Analysis of N. alba genome revealed genetic information similar to albonoursin biosynthetic gene cluster, albABC. An albABC gene deletion mutant of N. alba was generated. Liquid chromatography-mass spectrometry analysis showed that the mutant could not produce the cyclic dipeptides. Cyclic dipeptide production in the mutant was restored by genetic complementation with the albABC cloned in a native plasmid of Nocardiopsis. ß-Glucuronidase reporter assays with a second mutant construct, in which albABC promoter is transcriptionally fused to the reporting gene gusA, indicated that albABC gene expression was subject to osmoregulation. The system presented will be used to study the metabolic and genetic control of cyclic dipeptide biosynthesis in Nocardiopsis.

Bringing microbial interactions to light using imaging mass spectrometry.

Covering: up to the end of September 2013 Microorganisms are a plentiful resource for natural products research. Traditionally, natural products discovery from microbial sources depends on the screening of target-mediated inhibition. The natural products identified through this strategy usually correlate to significant microbial phenotypes. However, the target-mediated transcriptions deduced from low concentrations of natural products sometimes do not generate an obvious phenotype. The better understanding of the true biological roles of those microbial natural products will permit the application of rational approaches to the more effective exploitation of their use. Imaging mass spectrometry (IMS) has been developed and applied in many fields for decades. However, the applications of IMS on microbial natural products research have just been recently reported. IMS is one of few tools capable of revealing both phenotype and relevant and irrelevant chemotypes of microorganisms. In this review, we summarize the latest applications of IMS technologies. The challenges and prospect of improvement and application of IMS to microbial natural products research are discussed as well.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing.

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. BIOLOGICAL SIGNIFICANCE: Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Molecular and functional characterization of broccoli EMBRYONIC FLOWER 2 genes.

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.