Isolation of guard cell-enriched epidermal fragments and metabolite profiling analysis
Guard cell-enriched epidermal fragments (after simply called guard cells) were isolated as described earlier (Daloso et al., 2015). Guard cells were harvested at pre-dawn and in the early morning (120 min after sunrise) and used for metabolomics analysis. Metabolite extractions were performed using ~200 mg of guard cells. Polar metabolites were extracted using a well-established gas chromatography coupled to time of flight mass spectrometry (GC-TOF-MS) platform (Lisec et al., 2006). Chromatogram and mass spectral analyses were carried out using TagFinder software (Luedemann et al., 2008).
Metabolic network analysis
Correlation-based networks were created between relative guard cell metabolite content in dark and light for each genotype and between stomatal speediness parameters (Sl max and t50%) and relative guard cell metabolite changes in the dark and after the transition to the light using all genotypes data, in which the nodes correspond to the metabolites and the links to the strength of connection between nodes in module (positive or negative) by Pearson correlation. The relative guard cell metabolic changes were obtained by dividing the content of each metabolite in the lightper the average of those found in the dark within the genotype. The networks were designed by restricting the strength of the connections to a specific limit of Pearson correlation coefficient (r ) (-0.5 > r > 0.5). The network parameters, clustering coefficient, network heterogeneity, network density, network centralization and connected components were obtained as described in previous work (Assenov et al., 2008). Preferential attachment is a characteristic of scale-free networks in which as higher is the number of links of a node, higher is the probability of this node to receive new links (Albert & Barabási, 2002). Here, we determined the preferential attachment as the nodes that are considered as hub in the dark and that maintained the number of links in the light higher than the average of the network in the dark.