Introduction

It has been estimated that water demand for agricultural will increase ca. 17% by 2025, mostly due the increase in the average global temperature and the fact that drought episodes will become more frequent according to the predicted climate change scenarios (Dai, 2013; Pennisi, 2008; Rahmstorf & Coumou, 2011). It is thus important to improve plant water use efficiency (WUE), defined as the ratio between the amount of accumulated biomass per unit of water used or transpired (Condon et al., 2004). However, plant responses to adverse conditions are modulated by complex regulatory networks, which act at different spatial and temporal scales. This highlights the complexity of plant cell functioning and the difficulty in finding biotechnological targets for plant WUE improvement (Bertolli, Mazzafera & Souza, 2014). One important strategy to improve WUE is decreasing plant water consumption by genetic manipulation of key regulator(s) of stomatal movements (Flexas, 2016; Flütsch et al., 2020b; Gago et al., 2014; McAusland et al., 2016; Papanatsiou et al., 2019). The WUE fundamentally depends on the ratio between photosynthetic carbon assimilation and water lost by the transpiration process, it is reasonable to assume that stomata act as a master regulator of WUE (Brodribb, Sussmilch, & McAdam, 2019). However, although the stomatal development is relatively well understood (Dow & Bergmann, 2014; Qi & Torii, 2018), knowledge concerning the regulation of guard cell metabolism is insufficient, despite this being a great potential target for plant WUE improvement (Daloso et al., 2017; Gago et al., 2020; Lawson & Matthews, 2020).
Stomata are leaf epidermal structures consisting of two guard cells that surround a pore and, in certain cases, with additional subsidiary cells (Lima et al., 2018) whose aperture are actively regulated (Schroeder et al., 2001). Guard cells are highly specialized and integrate endogenous and environmental signals to regulate stomatal opening (Sussmilch, Schultz et al., 2019). Environmental cues such as temperature, soil water status, light, CO2 concentration and air vapor pressure deficit modulate stomatal movements in a mesophyll cells-dependent manner (Lawson et al., 2014; Mott, 2009). The dynamics of stomatal movements are thus closely linked to the mesophyll photosynthetic activity, in which the transport of mesophyll-derived metabolites such as sucrose and malate and their import into guard cells seem to be key for stomatal movement regulation (Daloso, dos Anjos, & Fernie, 2016; Gago et al., 2016; Lima et al., 2019; Wang et al., 2019). Indeed, genetic manipulation of genes regulating the trade-off between photosynthetic rate (A ) and stomatal conductance (g s) has been shown to be an effective strategy to improve photosynthesis, WUE and/or drought tolerance (Antunes et al., 2017; Araújo et al., 2011; Daloso et al., 2016b; Kelly et al., 2019; Laporte, Shen & Tarczynski, 2002; Lugassi et al., 2015; Nunes-Nesi et al., 2007). Guard cell genetic manipulation has been achieved through the use of guard cell specific promoters such as KST1 (Kelly et al., 2017; Kopka, Provart & Muller-Rober, 1997; Plesch, Ehrhardt & Mueller-Roeber, 2001), which is important to avoid undesired pleotropic modifications in mesophyll cells or sink tissues, notably when sugar-related genes are manipulated.
Several studies indicate the importance of carbohydrate metabolism for the regulation of stomatal movements (Daloso et al., 2016a; Granot & Kelly, 2019; Lima et al., 2018). It has been demonstrated that transgenic plants with modified guard cell sugar metabolism have altered stomatal movements. For instance, transgenic plants with increased expression of hexokinase or antisense inhibition of a sucrose transporter (SUT1) have increased WUE (Antunes et al., 2017; Kelly et al., 2019). By contrast, overexpression of sucrose synthase 3(StSUS3 ) increased g s, A and plant growth (Daloso et al., 2016b). Additionally, Arabidopsis plants lacking hexose transporters (STP1 and STP4) or enzymes related to starch degradation (AMY3 and BAM1) have altered guard cell sugar metabolism and reduced speed of light-induced stomatal opening (Flütsch et al., 2020a,b). These studies demonstrated that genetic manipulation of guard cell sucrose metabolism is a promising strategy to improve WUE. Furthermore, the role of sucrose in the regulation of stomatal movements has been reinterpreted on the basis of recent results. These include the demonstration that sucrose can induce stomatal closure in an ABA-mediated, hexokinase-dependent mechanism (Kelly et al., 2013; Lugassi et al., 2015), and that the degradation of sucrose within the guard cells is an important source of substrate for the TCA cycle and glutamine biosynthesis during light-induced stomatal opening (Daloso et al., 2015; Medeiros et al., 2018; Robaina-Estévez et al., 2017). Thus, guard cell sucrose metabolism seems to play a major role in regulating the A -g s trade-off during both stomatal opening and closure (Granot & Kelly, 2019; Lima et al., 2018).
Sucrose metabolism is not only important for the guard cell but also for the overall carbon distribution throughout the plant. In the cytosol of plant cells, sucrose is degraded into hexoses by different invertase (INV) and sucrose synthase (SUS) isoforms (Fettke and Fernie 2015). The number and the expression of SUS isoforms vary among plant species and organs (Angeles-Núñez & Tiessen, 2012; Baroja-Fernández et al., 2012; Bieniawska et al., 2007; Koch et al., 1992; Kopka et al., 1997). InNicotiana tabacum L., there are seven SUS isoforms (NtSUS1-7 ) and isoforms 2 and 3 are the most abundant in mature leaves (Wang et al., 2015). In Arabidopsis thaliana L., recent results indicate that AtSUS3 is solely expressed in embryo and guard cells (Yao, Gonzales-Vigil & Mansfield, 2020), similar to the expression pattern observed for its ortholog in Solanum tuberosumL. (Kopka et al., 1997). Furthermore, guard cell SUS activity is approximately 40-fold higher compared to that of whole leaves (Daloso et al., 2015). Taken together, these data suggest a central role of SUS in the regulation of guard cell metabolism and stomatal movements. Here we show that tobacco transgenic plants with mild reductions in guard cellNtSUS2 expression exhibited up to 44% less whole plant transpiration than wild type plants, yet only a minor impact on biomass production, corresponding to increased yield WUE (yWUE) in one of the transgenic lines under well-watered conditions. Surprisingly, the transgenic lines transpired more under water restriction periods, indicating a more efficient use of water under this condition. Our results are collectively discussed in terms of the role of NtSUS2 and guard cell sucrose metabolism in the regulation of stomatal movements and whole plant transpiration.