Guard cell NtSUS2 is important for the regulation of whole
plant transpiration
Sucrose has long been pointed out as an important metabolite that
regulates stomatal movements (Granot & Kelly, 2019; Talbott & Zeiger
1998). Previous studies from our group highlight that manipulating the
expression of StSUS3 alter g s, with slight
impacts on A , WPT and biomass production (Antunes et al., 2012;
Daloso et al., 2016b). Here, we generated tobacco transgenic plants
antisense for the StSUS3 under control of the guard cell specific
KST1 promoter. This led to reduced expression of ortholog NtSUS2in guard cell (Figure 1C) and decreased g s in the
transgenic lines (Figure 2E). These results are in agreement with those
observed in potato plants expressing an antisense construct targeted
against SUS3 under 35S promoter (Antunes et al., 2012). However,
it is important to highlight that the use of the constitutive 35S
promoter reduced A up to ~18%, whilst here the
KST1-mediated reduction in guard cell NtSUS2 expression have
slightly reduced g s with minor impact on A(~10% in average) under well-watered conditions. This
highlights the importance to use cell-specific promoters (Lawson et al.,
2014), especially when the genetic manipulation involves sucrose
metabolism, given its role in source-sink interaction.
Evidence linking sucrose metabolism and stomatal movement regulation
comes from several genetic reverse studies. Transgenic plants with
altered guard cell sucrolytic activity (Antunes et al., 2012; Daloso et
al., 2016b; Ni, 2012), increased expression of guard cell hexokinase
(Kelly et al., 2013, 2019; Lugassi et al., 2015), decreased expression
of a guard cell plasma membrane sucrose (SUT1 ) and hexose
(STP ) transporters (Antunes et al., 2017; Flütsch, Nigro et al.,
2020) or lacking key enzymes of starch breakdown (Flütsch et al., 2020b;
Horrer et al., 2016) have altered g s and/or
stomatal aperture. These studies collectively indicate that sugar
homeostasis and sucrose breakdown in guard cells are important to
sustain the energetic and metabolites demand of guard cells during
light-induced stomatal opening (Lawson & Matthews, 2020). They further
support the idea that sucrose breakdown and hexose phosphorylation
mediated by hexokinase is important during stomatal closure (Granot &
Kelly, 2019). Although it seems contradictory, it is important to
highlight that plants overexpressing hexokinase in guard cells can have
higher or lower E depending on the environmental conditions
(Lugassi et al., 2015). Similarly, the reductions in WPT observed in our
transgenic lines were more prominent under fluctuating environmental
(greenhouse) conditions, when compared to plants growing under
controlled (growth chamber) conditions. Furthermore, WPT ranges between
well-watered and water restriction periods, in which transgenic lines
transpired less under well-watered and tend to transpire more under
water shortage conditions. These results indicate that guard cell
sucrose metabolism is important to regulate stomatal movements according
to the prevailing environmental condition, highlighting the complexity
and the plasticity of guard cell metabolism in responding to changes in
the surrounding environment (Daloso et al., 2016a; Zeiger et al., 2002).
It is noteworthy that in the hottest and driest days of the experiment
(days 4, 5 and 6 – Figure 3A), both L3 and L13 lost up to 44% less
water than WT plants (Figures 3B,C). Furthermore, the L3 and L13
transgenic lines conserved 416 and 433 g H2O
plant-1 through eleven days, respectively (Figure 3).
Given that no changes in SD among the genotypes was observed (Figures
S5A), this indicates that the lower WPT is not associated to changes in
SD. Additionally, the WPT per leaf area remained significantly lower in
L3 than WT at the days 5, 8, 9 and 10 of the experiment, in which no
differences in leaf area between L3 and WT was observed at these days
(Figures 3E-G). This indicates that smallest leaf area was also not the
cause of the decreased WPT found in the transgenic lines. It seems
likely therefore that the lower WPT of the transgenic lines is
associated to the NtSUS2 -mediated guard cell metabolic changes,
as evidenced by both multivariate and metabolic network analyses
(discussed below).