Recent advances in proteomics and metabolomics in plants

Over the past decade,systems biology and plant-omics have increasingly become the main stream in plant biology research.New developments in mass spectrometry and bioinformatics tools,and methodological schema to inte-grate multi-omics data have leveraged recent advances in proteomics and metabolomics.These progresses are driv-ing a rapid evolution in the field of plant research,greatly facilitating our understanding of the mechanistic aspects of plant metabolisms and the interactions of plants with their external environment.Here,we review the recent progresses in MS-based proteomics and metabolomics tools and workflows with a special focus on their applications to plant biology research using several case studies related to mechanistic understanding of stress response,gene/protein function characterization,metabolic and signaling pathways exploration,and natural product discovery.We also present a projection concerning future perspectives in MS-based proteomics and metabolomics development including their applications to and challenges for system biology.This review is intended to provide readers with an overview of how advanced MS technology,and integrated application of proteomics and metabolomics can be used to advance plant system biology research.

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process

The soybean root system is complex.In addition to being composed of various cell types,the soybean root system includes the primary root,the lateral roots,and the nodule,an organ in which mutualistic symbiosis with N-fixing rhizobia occurs.A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont,resulting from the close cooperation between the plant cell and the bacteria.To date,the transcriptome of individual cells isolated from developing soybean nodules has been established,but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized.Using single-nucleus RNA-seq and Molecular Cartography technologies,we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B.diazoefficiens-infected cells in the mature soybean nodule,including those actively involved in nitrogen fixation and those engaged in senescence.Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process.For instance,we functionally characterized the role of GmFWL3,a plasma membrane microdomain-associated protein that controls rhizobial infection.Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

A novel expressed prostatic secretion(EPS)-urine metabolomic signature for the diagnosis of clinically significant prostate cancer

Objective:Significant efforts are currently being made to identify novel biomarkers for the diagnosis and risk stratification of prostate cancer(PCa).Metabolomics can be a very useful approach in biomarker discovery because metabolites are an important read-out of the disease when characterized in biological samples.We aimed to determine a metabolomic signature which can accurately distinguish men with clinically significant PCa from those affected by benign prostatic hyperplasia(BPH).Methods:We first performed untargeted metabolomics using ultrahigh-performance liquid chromatography tandem mass spectrometry on expressed prostatic secretion urine(EPS-urine)from 25 patients affected by BPH and 25 men with clinically significant PCa(defined as Gleason score≥3+4).Diagnosis was histologically confirmed after surgical treatment.The EPS-urine metabolomic approach was then applied to a larger,prospective cohort of 92 consecutive patients undergoing multiparametric magnetic resonance imaging for clinical suspicion of PCa prior to biopsy.Results:We established a novel metabolomic signature capable of accurately distinguishing PCa from benign tissue.A metabolomic signature was associated with clinically significant PCa in all subgroups of the Prostate Imaging Reporting and Data System(PI-RADS)classification(100%and 89.13%of accuracy when the PI-RADS was in range of 1–2 and 4–5,respectively,and 87.50%in the more critical cases when the PI-RADS was 3).Conclusions:A combination of metabolites and clinical variables can effectively help in identifying PCa patients that might be overlooked by current imaging technologies.Metabolites from EPS-urine should help in defining the diagnostic pathway of PCa,thus improving PCa detection and decreasing the number of unnecessary prostate biopsies.

Hands-on Workshops as An Efective Means of Learning Advanced Technologies Including Genomics,Proteomics and Bioinformatics

Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications： （1） immediate short-term training workshops and （2） long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute （NHLBI）-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health （NJH） and the University of Colorado School of Medicine （UCD）. The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consis- tently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.