In the past two decades, the post-genomic era envisaged high-throughput technologies, resulting in more species with available genome sequences. In-depth multi-omics approaches have evolved to integrate cellular processes at various levels into a systems biology knowledge base. Metabolomics plays a crucial role in molecular networking to bridge the gaps between genotypes and phenotypes. However, the greater complexity of metabolites with diverse chemical and physical properties has limited the advances in plant metabolomics. For several years, applications of liquid/gas chromatography (LC/GC)-mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been constantly developed. Recently, ion mobility spectrometry (IMS)-MS has shown utility in resolving isomeric and isobaric metabolites. Both MS and NMR combined metabolomics significantly increased the identification and quantification of metabolites in an untargeted and targeted manner. Thus, hyphenated metabolomics tools will narrow the gap between the number of metabolite features and the identified metabolites. Metabolites change in response to environmental conditions, including biotic and abiotic stress factors. The spatial distribution of metabolites across different organs, tissues, cells and cellular compartments is a trending research area in metabolomics. Herein, we review recent technological advancements in metabolomics and their applications in understanding plant stress biology and different levels of spatial organization. In addition, we discuss the opportunities and challenges in multiple stress interactions, multi-omics, and single-cell metabolomics.
Arabidopsis MAP
kinase 4 (MPK4) has been proposed
to be a negative player in plant immunity, and it is also activated
by pathogen-associated molecular patterns (PAMPs), such as flg22.
The molecular mechanisms by which MPK4 is activated and regulates
plant defense remain elusive. In this study, we investigated Arabidopsis defense against a bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000
when Brassica napus MPK4 (BnMPK4) is overexpressed. We showed an increase in pathogen resistance
and suppression of jasmonic acid (JA) signaling in the BnMPK4 overexpressing (OE) plants. We also showed that the OE plants have
increased sensitivity to flg22-triggered reactive oxygen species (ROS)
burst in guard cells, which resulted in enhanced stomatal closure
compared to wild-type (WT). During flg22 activation, dynamic phosphorylation
events within and outside of the conserved TEY activation loop were
observed. To elucidate how BnMPK4 functions during
the defense response, we used immunoprecipitation coupled with mass
spectrometry (IP-MS) to identify BnMPK4 interacting
proteins in the absence and presence of flg22. Quantitative proteomic
analysis revealed a shift in the MPK4-associated protein network,
providing insight into the molecular functions of MPK4 at the systems
level.
Glycosylphosphatidylinositol (GPI) anchorage of cell
surface proteins
to the membrane is biologically important and ubiquitous in eukaryotes.
However, GPIs do not contain long enough lipids to span the entire
membrane bilayer. To transduce binding signals, GPIs must interact
with other membrane components, but such interactions are difficult
to define. Here, a new method was developed to explore GPI-interacting
membrane proteins in live cell with a bifunctional analogue of the
glucosaminylphosphatidylinositol motif conserved in all GPIs as a
probe. This probe contained a diazirine functionality in the lipid
and an alkynyl group on the glucosamine residue to respectively facilitate
the cross-linkage of GPI-binding membrane proteins with the probe
upon photoactivation and then the installation of biotin to the cross-linked
proteins via a click reaction for affinity-based protein isolation
and analysis. Profiling the proteins pulled down from the Hela cells
revealed 94 unique and 18 overrepresented proteins compared to the
control, and most of them are membrane proteins and many are GPI-related.
The results have proved not only the concept of using the new bifunctional
GPI probe to investigate GPI-binding membrane proteins but also the
important role of inositol in the biological functions of GPI anchors
and GPI-anchored proteins.
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