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After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific molecular mechanisms remain elusive. The lipid transfer protein defective in induced resistance 1 (DIR1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Here, we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1-1 mutant during SAR. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were also changed in guard cells of dir1-1 compared to the wild type. Identification of guard cell-specific SAR-related molecules may lead to new avenues of genetic modification/molecular breeding for disease-resistant plants. 

Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics, and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a biphasic fractionation method to extract proteins, metabolites, and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites, and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes, and allow quantitative analyses of different molecules and pathways. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility, and tight correlation between different types of biomolecules. 

Systemic Acquired Resistance (SAR) improves immunity of plant systemic tissue after local exposure to a pathogen. Guard cells that form stomatal pores on leaf surfaces recognize bacterial pathogens via pattern recognition receptors, such as Flagellin Sensitive 2 (FLS2). However, how SAR affects stomatal immunity is not known. In this study, we aim to reveal molecular mechanisms underlying the guard cell response to SAR using multi-omics of proteins, metabolites and lipids. Arabidopsis plants previously exposed to pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when they are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreased number of bacteria in leaves. Multi-omics has revealed molecular components of SAR response specific to guard cells functions, including potential roles of reactive oxygen species (ROS) and fatty acid signaling. Our results show an increase in palmitic acid and its derivative in the primed guard cells. Palmitic acid may play a role as an activator of FLS2, which initiates stomatal immune response. Improved understanding of how SAR signals affect stomatal immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.