On the role of guard cell metabolism for stomatal
speediness regulation
The speediness of stomatal responses to environmental cues has recently
received considerable attention to understand both the evolutionary
origin of active stomatal control and its potential to maximize plant
growth and WUE (Brodribb & McAdam, 2017; Flütsch et al., 2020a,b; Lima
et al., 2019; Papanatsiou et al., 2019; Sussmilch, Roelfsema & Hedrich,
2019). However, the mechanisms regulating stomatal speediness remain far
from clear (Lawson & Vialet-Chabrand, 2019). It is known that the
degradation of starch, sucrose and lipids are important during
light-induced stomatal opening (Daloso et al., 2015; Daloso et al.,
2016b; Horrer et al., 2016; McLachlan et al., 2016). Given the role of
malate as a counter ion of K+ during light-induced
stomatal opening, it was long hypothesized that malate would be the fate
of the carbon released from starch breakdown (Horrer et al. 2016;
Lasceve, Leymarie & Vavasseur, 1997; Outlaw & Manchester, 1979;
Schnabl, 1980; Schnabl, Elbert & Krämer, 1982; Talbott & Zeiger,
1993). However, recent evidence indicates that starch degradation and
sugar homeostasis within guard cells are key to speed up light stomatal
responses, but this was not associated to a differential malate
accumulation among WT and amy3 bam1 double mutant (Flütsch et
al., 2020b). Additionally, no 13C enrichment in
malate, fumarate and succinate was observed in guard cells under13C-sucrose feeding during dark-to-light transition,
but a substantial part of the 13C released from13C-sucrose was incorporated into Gln under this
condition (Medeiros et al., 2018). It seems likely therefore that the
glycolytic fluxes are not directed to malate synthesis, as long
hypothesized. In agreement with this idea, malate was not correlated to
stomatal speediness and did not appear in the S-plots or VIP score lists
here, despite the differences found in g s,
stomatal speediness and WPT between WT and the transgenic lines. By
contrast, other TCA cycle related metabolites such as citrate,
aconitate, Asp, Glu and Pro were positively associated to stomatal
speediness (Figure 8A) and citrate, aconitate, isocitrate and fumarate
were included in the S-plots and the VIP score lists of at least one
transgenic line (Figures 6 and S5).Furthermore, these metabolites have
higher relative level in the WT when compared to both transgenic lines
after dark-to-light transition (Figures 6 and S5), suggesting a higher
activation of the TCA cycle and associated amino acid biosynthetic
pathways in WT, which may contribute to explain the fast light stomatal
response in this genotype.
Our results collectively further indicate that the decreased guard cellNtSUS2 expression reduced the amount of substrate for glycolysis,
which, in turn, affected the synthesis of amino acids. This idea is
supported by the multivariate analyses in which Ala and Ser have lower
accumulation in the transgenics and were included in both S-plots and
VIP score lists (Figures 6 and S5). Furthermore, six amino acids (Ala,
Ser, Asp, Glu, Ile and Pro) were positively correlated with stomatal
speediness (Figure 8A). In contrast, trehalose and adipic acid were
negatively associated to stomatal speediness (Figure 8A) and have higher
relative level in the transgenics than the WT after dark-to-light
transition (Figure 8B). Whilst information concerning the role of adipic
acid in plant metabolism is scarce (Rodgman & Perfetti, 2013),
trehalose metabolism has been closely associated to the regulation of
stomatal movements (Daloso et al., 2016a; Figueroa & Lunn, 2016; Lunn
et al., 2014). Although the exact mechanism by which trehalose
metabolism contribute to regulate stomatal movements remain unclear,
recent findings indicate that sugar homeostasis in guard cells is
important to speed up light stomatal responses (Flütsch et al., 2020a,b)
and to maximize the rapid early morning increase ing s (Antunes et al., 2017). Thus, given that
trehalose and trehalose-6-phosphate are closely associated to starch,
sucrose, organic acids and amino acids metabolisms (dos Anjos et al.,
2018; Figueroa et al., 2016; Martins et al., 2013), the differential
accumulation of trehalose in the transgenics suggests that sugar
homeostasis has been at least partially compromised in the transgenics.
This idea is further supported by the fact that several sugars such as
allose, idose, maltose, glucose, psicose and fructose were found in
S-plots or VIP score lists in at least one transgenic line (Figures
6C,D; S5C,D). Sucrose was positively correlated with stomatal speediness
(Figure 8A). Given that SUS works on both sucrose synthesis and
degradation directions, this reinforce the idea that sugar homeostasis
has been compromised in the transgenic lines, which in turn contributed
to reduce the synthesis of organic acids and amino acids and the
stomatal speediness in the transgenics in the light.
Our metabolic network analysis indicates that mild reductions inNtSUS2 expression substantially alter both the topology and the
connectivity of the guard cell metabolic network during dark-to-light
transition. It seems likely therefore that NtSUS2 is an important
regulator of guard cell metabolism, highlighting why this gene is highly
expressed in guard cells, when compared to mesophyll cells (Bates et
al., 2012; Bauer et al., 2013; Yao et al., 2020). Given the complexity
of guard cell metabolism, instead of using the classical reductionism
perspective of searching for particular genes, proteins and metabolites
that regulate stomatal speediness, a systemic view of the guard cell
metabolic changes during dark-to-light transition might offer better
possibilities to fully comprehend the regulation of stomatal kinetics
(Medeiros et al., 2015). For this, incorporating dynamic metabolic data
into genome scale metabolic models will certainly contribute to improve
our understanding on the metabolism-mediated stomatal kinetics
regulation. However, it is still unclear what are the key regulatory
points in the guard cell metabolic fluxes. Evidence suggests that the
fluxes toward glycolysis and the TCA cycle differ substantially in guard
cells compared to mesophyll cells in the light (Hedrich, Raschke &
Stitt, 1985; Horrer et al., 2016; Medeiros et al., 2018; Robaina-Estévez
et al., 2017; Zhao & Assmann, 2011). This probably involves a different
regulation of key glycolytic enzymes, which collectively may coordinate
the fluxes from starch and sucrose breakdown toward the TCA cycle and
associated pathways. However, diffuse information regarding the
mechanisms that regulate guard cell metabolism hamper our understanding
on the regulation of stomatal kinetics. Furthermore, guard cell
metabolism complexity is often underestimated based on analogies made
with mesophyll cells. However, guard cells have more than 1,000 genes
that are differentially expressed with respect to mesophyll cells and
seems to have higher plasticity in adjusting its metabolism according to
soil water availability, light quality and intensity,
CO2 concentration, air humidity and several others
endogenous and environmental cues.