Mild reductions in guard cell sucrose synthase 2 expression leads to slower stomatal opening and decreased whole plant transpiration in Nicotiana tabacum L

https://doi.org/10.1016/j.envexpbot.2020.104370Get rights and content

Highlights

  • NtSUS2-silenced plants have slower light-induced stomatal opening.

  • NtSUS2-silenced plants have a strong reduction in whole plant transpiration.

  • NtSUS2 silencing altered guard cell metabolic network.

  • NtSUS2 silencing reduce the accumulation of metabolites positively correlated with stomatal speediness.

  • The transgenic lines have higher effective use of water under short water deficit periods.

Abstract

The understanding of the dynamics of stomatal movements has increased substantially through genetic manipulation of plant metabolism either at the whole plant level or specifically in guard cells. However, the regulation of stomatal speediness remains not completely elucidated. Here we shown that reduced expression of guard cell sucrose synthase 2 (NtSUS2) of Nicotiana tabacum L. altered the topology and the connectivity of the guard cell metabolic network and the accumulation of metabolites positively correlated with stomatal speediness during dark-to-light transition. This leads to a slower light-induced stomatal opening, lower steady-state stomatal conductance and a strong reduction (up to 44 %) in daily whole plant transpiration in the transgenics, when compared to wild type plants. Furthermore, the transgenic lines transpired more or have lower reduction in whole plant transpiration under short water deficit periods, indicating a higher effective use of water under this condition. Our results collectively suggest that the regulation of stomatal movement and speediness involve a complex modulation of the guard cell metabolic network, in which NtSUS2 has an important role. The results are discussed on the role of guard cell metabolism for the regulation of both stomatal speediness and whole plant transpiration.

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 and 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 et al., 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 et al., 2019). However, although the stomatal development is relatively well understood (Dow and Bergmann, 2014; Qi and 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 and 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 et al., 2019b). 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 et al., 2016a; 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 (gs) 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 et al., 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 et al., 1997; Plesch et al., 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 and 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 gs, 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, 2020b). 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-gs trade-off during both stomatal opening and closure (Granot and 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 and Tiessen, 2012; Baroja-Fernández et al., 2012; Bieniawska et al., 2007; Koch et al., 1992; Kopka et al., 1997). In Nicotiana 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 et al., 2020), similar to the expression pattern observed for its ortholog in Solanum tuberosum L. (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. We thus hypothesize that silencing SUS specifically in guard cells would reduce gs and possibly improve WUE. Here we show that tobacco transgenic plants with mild reductions in guard cell NtSUS2 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.

Section snippets

Plant material and growth conditions

Nicotiana tabacum L. cv Havana 425 wild type (WT) and transgenic plants in which the sucrose synthase 2 (NtSUS2) gene expression was suppressed under the control of the KST1 promoter (X79779). The transformation was carried out by cloning the SUS3 gene from Solanum tuberosum (StSUS3) (STU24088) into the pBinK plasmid vector, which was derived from pBinAR-Kan (Höfgen and Willmitzer, 1990) but had the CaMV-35S promoter replaced by the KST1 promoter. A 1567 bp fragment was obtained by the

Phylogenetic analysis indicates that NtSUS2 is ortholog of StSUS3

We have previously cloned potato sucrose synthase 3 (StSUS3) and inserted in the sense direction into tobacco guard cells in order to investigate the function of this gene in guard cell metabolism and stomatal movements (Daloso et al., 2016b). The reasons to use StSUS3 and tobacco plants are based in the fact that StSUS3 and its ortholog AtSUS3 has been previously shown to be highly expressed in guard cells (Bates et al., 2012; Bauer et al., 2013; Kopka et al., 1997; Yao et al., 2020) and that

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 and Kelly, 2019; Talbott and Zeiger, 1998). Previous studies from our group highlight that manipulating the expression of StSUS3 alter gs, 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

Conclusions

Tobacco transgenic plants with antisense construction target to guard cell NtSUS2 had slower stomatal opening in the light and decreased steady-state gs and WPT under well-watered conditions. Furthermore, the transgenic lines showed lower reduction or higher WPT under short water restriction periods, indicating a greater effective use of water under these conditions. Our results provide further evidence that NtSUS2 is an important regulator of guard cell metabolic network and strengthen the

Funding

This work was supported by the National Council for Scientific and Technological Development (CNPq) [grant number 428192/2018-1].

Accession numbers

Sequence from StSUS3 and NtSUS1-7 genes data used on this article can be found in the National Center for Biotechnology Information databases (https://www.ncbi.nlm.nih.gov/) under accession numbers: AY205084.1, XM_016618980.1, XM_016595403.1, XM_016627888.1, XM_016610900.1, XM_016608012.1, XM_016585183.1, XM_016611295.1, respectively.

Author contributions

F.B.S.F. and D.M.D. designed the research and experiments; F.B.S.F., R.L.G.B., R.S.C.B., S.A.C. and D.B.M. performed the experiments; all authors contributed to write the final manuscript; D.M.D. obtained funding and is responsible for this article.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was made possible through financial support from the National Council for Scientific and Technological Development (CNPq, Grant 428192/2018-1). We also thank the research fellowship granted by CNPq to D.M.D. and the scholarships granted by CNPq to F.B.S.F. and R.L.G.B. and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES-Brazil) to R.S.C.B. and S.A.C.

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