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).