Updated project metadata. In order to discover new nodes and edges for low CO2-induced stomata closure, we applied hyphenated mass spectrometry (MS)-based metabolomics and proteomics approaches to analyze short-term low CO2 responses in B. napus (canola) guard cells. A total of 411 metabolites were quantified. We observed decreased trends of primary metabolites biosynthesis (e.g., most common amino acids, nucleotides, and sugars), and changes in the levels of osmoregulators such as sucrose, malate, and mannitol showed increased trends at both early and late time points. Unlike under elevated CO2 levels, JA biosynthesis was not altered. Instead, phytohormones that induces stomatal opening, including cytokinins, auxins, GA4, as well as melatonin increased at early time points. This study also highlights the utility of single cell-type metabolomics in discovering and testing new nodes and edges in cellular signaling and metabolic networks.