3.1 The changes in metabolite content were similar between
dark-exposed and illuminated guard cells
Overall, the metabolic changes were similar between dark-exposed and
illuminated guard cells. Under both dark and light conditions, the
accumulation of fumarate, pyruvate, aspartate and lactate and the
degradation of sucrose, glucose, gamma-aminobutyric acid (GABA) and
different amino acids was observed (Figure 1). However, also
differential behaviour of specific metabolites was observed. After 60
min of illumination the content of both glutamate (Glu) and urea
increased as compared to the time 0 of the experiment and fructose,
sorbose and citrate levels decreased (Figure 1). Comparing dark and
light treatments in each time point, decreased content of alanine,
citrate, glucose, myo -inositol, sorbose and sucrose was observed
in illuminated samples. Interestingly, the content of malate is lower at
10 and 60 min and higher at 20 min in the light, as compared to
dark-exposed guard cells (Supplemental Figure S1). This analysis
highlights that the dynamic of relative metabolite
accumulation/degradation throughout time is slightly altered by light
imposition. This idea is further supported by principal component
analysis (PCA), in which no clear separation of dark and light
treatments was observed (Figure 2).
3.2 Illumination
triggers re-modelling of metabolic network topology in guard cells
We have recently shown that light-induced stomatal opening involves
changes in the density and topology of the guard cell metabolic network
(Freire et al. 2021). Here, no general pattern of increasing or
decreasing network parameters such as clustering coefficient,
centralization, density and heterogeneity was observed over time in
metabolic networks created using metabolite content data (Table S1).
However, the number of hub-like nodes, the preferential attachment and
the appearance of hub-like nodes differ between dark and light samples.
Whilst these parameters reduced to zero after 60 min in the dark, they
increased in the light metabolic network over time (Table S1), in which
malate, fumarate, GABA and glucose appear as important hubs of the
metabolic network of illuminated guard cells (Figure 3). These results
suggest that illumination alter the metabolic network structure of guard
cells, with particularly strong impact on the pathways associated to the
TCA cycle and sugar metabolism.
3.3 Guard cells
have a highly active metabolism in the dark
Similar to the described for metabolite content data, dark-exposed and
illuminated guard cells display similar trends in the relative13C-enrichment (R13C) data. Alanine,
fumarate, glucose, glycine, sucrose and valine were labelled with
statistical significance (P < 0.05) under both
conditions in at least one time point, as compared to the time 0 of the
experiment (Figure 4). However, comparing light and dark treatments in
each time point, increased 13C-labelling in sucrose,
fructose, sorbose, malate, pyruvate, succinate and glutamate was
observed in illuminated guard cells, especially at 60 min of labelling
(Supplemental Figure S2). In fact, PCA indicates that dark and light
treatments differ mainly at 60 min, as evidenced by the separation by
the first component (Figure 5). Analysis of the contribution of each
metabolite for the PCA highlights that certain sugars (sucrose, fructose
and glucose), organic acids (citrate and pyruvate), amino acids (Glu,
Ile) and myo -inositol are those that mostly contributed to the
separation observed in PCA of 60 min (Supplemental Figure S3). These
results indicate that several metabolic pathways are activated following
PEPc-mediated CO2 assimilation in the dark, including
gluconeogenesis and the TCA cycle. However, the combination of
CO2 assimilation catalysed by both PEPc and RuBisCO
leads to more dramatic changes in 13C-distribution
over these and other metabolic pathways in illuminated guard cells.