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Stomatal immunity is the primary gate of the plant pathogen defense system. Non-expressor of Pathogenesis Related 1 (NPR1) is the salicylic acid (SA) receptor, which is critical for stomatal defense. SA induces stomatal closure, but the specific role of NPR1 in guard cells and its contribution to systemic acquired resistance (SAR) remain largely unknown. In this study, we compared the response to pathogen attack in wild-type Arabidopsis and the npr1-1 knockout mutant in terms of stomatal movement and proteomic changes. We found that NPR1 does not regulate stomatal density, but the npr1-1 mutant failed to close stomata when under pathogen attack, resulting in more pathogens entering the leaves. Moreover, the ROS levels in the npr1-1 mutant were higher than in the wild type, and several proteins involved in carbon fixation, oxidative phosphorylation, glycolysis, and glutathione metabolism were differentially changed in abundance. Our findings suggest that mobile SAR signals alter stomatal immune response possibly by initiating ROS burst, and the npr1-1 mutant has an alternative priming effect through translational regulation. 

Large-scale high throughput metabolomic technologies are indispensable components of systems biology in terms of discovering and defining the metabolite parts of the system. However, the lack of a plant metabolite spectral library limits the metabolite identification of plant metabolomic studies. Here, we have created a plant metabolite spectral library using 544 authentic standards, which increased the efficiency of identification for untargeted metabolomic studies. The process of creating the spectral library was described, and the mzVault library was deposited in the public repository for free download. Furthermore, based on the spectral library, we describe a process of creating a pseudo-targeted method, which was applied to a proof-of-concept study of Arabidopsis leaf extracts. As authentic standards become available, more metabolite spectra can be easily incorporated into the spectral library to improve the mzVault package. 

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. 

The primary goal of this study was to determine the effect of sonication on stomatal movement. A minor goal was to determine the best time interval at which sonication is the most effective at removing mesophyll cells and enriching guard cells. For this study, abaxial leaf peels of Arabidopsis thaliana were sonicated for 1, 3, 5, and 7-minute intervals at a set amplitude to analyze the removal of mesophyll cells. To juxtapose the leaves and to determine guard cell enrichment, microscopic images were taken prior to and after sonication. Furthermore, to establish that the stomata are alive, neutral red staining was used in conjunct with 40x magnification. It was hypothesized that sonication is an effective method for the removal of mesophyll cells and enrichment of guard cells. The results of this study suggest that sonication is in fact an effective protocol for guard cell enrichment; however, it is not as effective for guard cell purification. This is due to the presence of mesophyll cells and epidermal layers present after sonication. Previous research dealing with sonication is very prevalent; however, research on sonication dealing with the removal of mesophyll cells in A. thaliana is not widely studied. Thus, previous information to support this study could not be attained. Results from the first part of the experiment were then extended to determine how sonication affects stomatal movement. It was determined that in the experimental group, the average stomatal aperture decreased over a two-hour period.