Abstract
Drought can severely damage crops, resulting in major yield losses. During drought, vascular land plants conserve water via stomatal closure. Each stomate is bordered by a pair of guard cells that shrink in response to drought and the associated hormone abscisic acid (ABA). The activation of complex intracellular signaling networks underlies these responses. Therefore, analysis of guard cell metabolites is fundamental for elucidation of guard cell signaling pathways. Brassica napus is an important oilseed crop for human consumption and biodiesel production. Here, non-targeted metabolomics utilizing gas chromatography mass spectrometry (GC-MS/MS) and liquid chromatography mass spectrometry (LC-MS/MS) were employed for the first time to identify metabolic signatures in response to ABA in B. napus guard cell protoplasts. Metabolome profiling identified 390 distinct metabolites in B. napus guard cells, falling into diverse classes. Of these, 77 metabolites, comprising both primary and secondary metabolites were found to be significantly ABA responsive, including carbohydrates, fatty acids, glucosinolates, and flavonoids. Selected secondary metabolites, sinigrin, quercetin, campesterol, and sitosterol, were confirmed to regulate stomatal closure in Arabidopsis thaliana, B. napus or both species. Information derived from metabolite datasets can provide a blueprint for improvement of water use efficiency and drought tolerance in crops.
Highlights
Plants are sessile organisms that are continuously subjected during their lifecycles to a spectrum of environmental signals and stimuli, including both abiotic factors such as availability of water, light, and nutrients, and biotic factors such as interactions with both beneficial and pathogenic organisms
We first confirmed that 10 μM abscisic acid (ABA), a concentration typically used in assays of stomatal responses[29,30,31], is sufficient to induce stomatal closure in both leaf pieces (Fig. 1A) and epidermal peels (Fig. 1A) of B. napus line DH12075
These results indicate the effectiveness of the ABA concentration used for our subsequent metabolomics analyses on B. napus guard cell protoplasts (GCPs)
Summary
Plants are sessile organisms that are continuously subjected during their lifecycles to a spectrum of environmental signals and stimuli, including both abiotic factors such as availability of water, light, and nutrients, and biotic factors such as interactions with both beneficial and pathogenic organisms. Using an iTRAQ (isobaric tag for relative and absolute quantitation)-based comparative proteomics approach, 66 and 38 proteins were found to be significantly induced and suppressed by ABA in B. napus guard cells, respectively These ABA responsive proteins participate in photosynthesis, metabolism, energy, protein synthesis, stress/defense (antioxidant system and glucosinolate-myrosinase system), membrane and transport processes, and protein folding/transport www.nature.com/scientificreports/. One landmark application of metabolomics to study the stress regulated metabolome at the level of the single cell type was an investigation of the ABA responsive metabolic changes in guard cell protoplasts from A. thaliana wild type and heterotrimeric G-protein α subunit mutant, gpa[1], using targeted metabolomics with multiple reaction monitoring (MRM)[12]. Interaction with other hormones, indole-3-acetic acid (IAA), in ABA modulated stomatal movement was revealed, validating phytohormone crosstalk[12] These targeted MRM-based profiles of the A. thaliana guard cell metabolome provided the first example of investigating dynamic metabolome changes of a single-cell-type in plants
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