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.