Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth

Article6 July 2020Open Access Transparent process Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth Sabrina Flütsch orcid.org/0000-0001-7020-6520 Institute of Integrative Biology, ETH Zürich, Zürich, Switzerland Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Arianna Nigro Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Franco Conci Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Jiří Fajkus Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Matthias Thalmann orcid.org/0000-0002-6260-1448 Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Martin Trtílek Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Klára Panzarová Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Diana Santelia Corresponding Author [email protected] orcid.org/0000-0001-9686-1216 Institute of Integrative Biology, ETH Zürich, Zürich, Switzerland Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Sabrina Flütsch orcid.org/0000-0001-7020-6520 Institute of Integrative Biology, ETH Zürich, Zürich, Switzerland Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Arianna Nigro Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Franco Conci Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Jiří Fajkus Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Matthias Thalmann orcid.org/0000-0002-6260-1448 Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Martin Trtílek Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Klára Panzarová Photon Systems Instruments (PSI), Drasov, Czech Republic Search for more papers by this author Diana Santelia Corresponding Author [email protected] orcid.org/0000-0001-9686-1216 Institute of Integrative Biology, ETH Zürich, Zürich, Switzerland Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland Search for more papers by this author Author Information Sabrina Flütsch1,2,‡, Arianna Nigro2,†,‡, Franco Conci2, Jiří Fajkus3, Matthias Thalmann2,†, Martin Trtílek3, Klára Panzarová3 and Diana Santelia *,1,2 1Institute of Integrative Biology, ETH Zürich, Zürich, Switzerland 2Department of Plant and Microbial Biology, University of Zürich, Zürich, Switzerland 3Photon Systems Instruments (PSI), Drasov, Czech Republic †Present address: Syngenta Crop Protection AG, Stein AG, Switzerland †Present address: John Innes Centre, Norwich Research Park, Norwich, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +41 44 632 89 27; E-mail: [email protected] EMBO Rep (2020)21:e49719https://doi.org/10.15252/embr.201949719 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis. Synopsis This study uncovers a new role for plasma membrane Sugar Transport Protein 1 and 4 (STP1/STP4) in glucose uptake to Arabidopsis thaliana guard cells, which is essential for stomatal movements and plant growth. Light-induced stomatal opening is inhibited in stp1stp4 double mutants, severely impairing CO2 assimilation and biomass production, which is reversed by elevated ambient-air CO2 concentrations. stp1stp4 guard cells have almost undetectable amounts of glucose after 40 min of light and remain flaccid. stp1stp4 guard cells are devoid of starch. Micro-grafting experiments further demonstrate that the stp1stp4 stomatal phenotype is not linked to the function of these transporters in the roots. Introduction Stomata are microscopic pores on the plant leaf epidermis surrounded by a pair of guard cells. These vital cells adjust pore aperture in response to numerous endogenous and exogenous factors, allowing uptake of carbon dioxide (CO2) for photosynthesis (A), while preventing excessive water loss through transpiration (E). By controlling the trade-off between photosynthesis and transpiration, stomata play a critical role in determining water-use efficiency (WUE = amount of carbon fixed per unit water loss, A/E) and, hence, plant growth and productivity (Lawson & Vialet-Chabrand, 2019). Stomatal opening and closure results from reversible changes in guard cell volume and shape. At the molecular level, this is driven primarily by the activity of the plasma membrane H+-ATPase which stimulates the movement of large quantities of ions (mainly potassium, chloride, malate2-, and nitrate) into and out of the guard cells and consequent osmotic water flow for their swelling or shrinking (Inoue & Kinoshita, 2017; Jezek & Blatt, 2017). For more than a century, it has been known that guard cells possess modified carbohydrate metabolic pathways compared to the rest of the leaf, but their significance for stomatal function has long remained obscure. Only recently it became clear that guard cell starch metabolism integrates with signaling and membrane ion transport to regulate stomatal movements (Daloso et al, 2017; Santelia & Lawson, 2016). At the start of the day, the rapid breakdown of guard cell starch is activated by phototropin-mediated signaling downstream of the plasma membrane H+-ATPase to promote efficient stomatal opening (Horrer et al, 2016). The major guard cell starch-derived metabolite is glucose (Glc), which is needed to maintain the cytoplasmic sugar pool contributing to fast stomatal opening (Flütsch et al, 2020). Starch formation induced during high CO2-mediated stomatal closure has been proposed to facilitate the dissipation of the accumulated organic solutes leading to changes in guard cell osmotic potential for water efflux (Penfield et al, 2012; Azoulay-Shemer et al, 2016). Soluble sugars also regulate stomatal movements, but the way they do so remains still rather controversial (Daloso et al, 2016a). Initial studies suggested that sucrose (Suc) and its derived sugars (Glc; fructose, Fru) induce stomatal opening as direct osmotica (Outlaw & Manchester, 1979; Poffenroth et al, 1992; Talbott & Zeiger, 1993; Amodeo et al, 1996). More recently, it was revealed that Suc promotes stomatal opening by serving as a substrate for glycolysis and mitochondrial respiration (Daloso et al, 2015, 2016b; Medeiros et al, 2018). Sugars can also induce stomatal closure either as osmolytes in the guard cell apoplast (Lu et al, 1997; Outlaw & De Vlieghere-He, 2001; Kang et al, 2007) or as signaling molecules through phosphorylation by hexokinase within the guard cells (Kelly et al, 2013; Lugassi et al, 2015). Despite these studies shed light on the importance of carbohydrate metabolism for stomatal movements, experiments have not yet answered basic questions about the source of sugars in guard cells. Given that CO2 fixation within guard cells is limited (Outlaw et al, 1979; Outlaw, 1989; Reckmann et al, 1990) and that photosynthesis in the mesophyll cells is the main source of sugars at the whole-plant level, it is likely that symplastically isolated guard cells rely mostly on mesophyll-derived Suc to fulfill their metabolic needs. Suc can be taken up directly via Suc transporters or in the form of hexoses via monosaccharide transporters following Suc hydrolysis by a cell wall invertase. Transcriptomics studies suggest that several sugar transporters are highly expressed in guard cells (Leonhardt et al, 2004; Wang et al, 2011; Bates et al, 2012; Bauer et al, 2013), but the relative contribution of this transport system to guard cell function is not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for Glc import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight that a tight coordination between mesophyll and guard cell carbohydrate metabolism promotes optimal plant growth through regulation of stomatal opening. Results Sugar Transport Protein 1, 4, and 13 are highly expressed in guard cells Higher plants possess three types of plasma membrane carriers for the intercellular transport of sugars: MSTs (monosaccharide transporters), SUCs or SUTs (Suc transporters) and SWEETs (hexose and Suc transporters). Angiosperm genomes usually contain several paralogs of each class of transporters, most of which serve distinct physiological roles (Chen et al, 2015). Through literature and database searches, we identified 40 plasma membrane sugar transporters in the Arabidopsis genome, covering all three types of carriers (Appendix Table S1). To select potential candidates for our study, we performed in silico analysis of gene expression levels in Arabidopsis guard cells using publicly available expression data (Fig EV1A). As expected, several transporters were highly expressed in guard cells, for instance, sucrose transporters 1, 2, and 3 (SUC1, SUC2, SUC3); Sugars will eventually be exported transporters 1, 5, 11, and 12 (SWEET1, SWEET5, SWEET11, SWEET12); sugar transport proteins 1, 4, 5, and 13 (STP1, STP4, STP5, STP13); and polyol/monosaccharide transporters 4, 5 and 6 (PMT4, PMT5, PMT6) (Fig EV1A). We focused on STP1, 4, and 13, as their gene expression in guard cells was on average 15 to 40 times higher compared to other sugar transporters (Fig EV1A). We confirmed their high and preferential expression in guard cell-enriched epidermal peels by qPCR (Fig EV1B and Appendix Table S2). In a previous study, STP1 was shown by in situ hybridization and immunohistochemistry to localize to guard cells (Stadler et al, 2003), further supporting our results. Click here to expand this figure. Figure EV1. STP1, STP4 and STP13 are highly and preferentially expressed in guard cells In silico analysis of plasma membrane sugar transporter gene expression levels in Arabidopsis guard cells. Arabidopsis eFP browser (http://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html); Arabidopsis guard cell protoplasts (Yang et al, 2008). STP1, STP4, and STP13 gene transcript levels in WT guard cell-enriched epidermal peels compared to WT rosette leaves at the end of the night. KAT1 and MYB60 were used as guard cell-specific markers, whereas BAM3 was used as leaf-specific marker. Data for two independent experiments are shown; means ± fold change range n ≥ 6. STP1, STP4, and STP13 gene transcript levels in WT rosette leaves compared to stp1-1, stp4-1, and stp13 rosette leaves at the end of the night. Data for two independent experiments are shown; means ± fold change range n ≥ 6. STP1 and STP4 gene transcript levels in WT rosette leaves compared to stp1-2 and stp4-2 rosette leaves at the end of the night. Data for two independent experiments are shown; means ± fold change range n ≥ 5. Data information: (B, C, and D) ACT2 was used as a housekeeping gene for normalization. For details about fold change and error calculations, see Materials and Methods section. Primer sequences and efficiencies are given in Appendix Table S2. Download figure Download PowerPoint STPs are high-affinity monosaccharide/proton symporters responsible for the transport of Glc, Fru, galactose, mannose, arabinose, and xylose from the apoplastic space into the cytosol (Büttner & Sauer, 2000; Büttner, 2010; Poschet et al, 2010; Rottmann et al, 2016, 2018b). These transporters are mostly found in sink tissues or symplastically isolated cells, such as pollen tubes, developing embryo, or guard cells (Stadler et al, 2003; Büttner, 2010; Rottmann et al, 2018a). It has been shown that STPs fulfill three main functions in plants: uptake of monosaccharides for the nutrition of sink cells (Sherson et al, 2000); re-absorption of monosaccharides from damaged roots under abiotic stress (Yamada et al, 2011); and antibacterial defense by competing with pathogens for extracellular sugars (Yamada et al, 2016). Light-induced stomatal opening is impaired in Arabidopsis plants lacking both STP1 and STP4 transporters To assess the contribution of the selected STPs to stomatal function, we obtained homozygous Arabidopsis T-DNA insertion lines at the STP1 (stp1-1; SALK_048848 and stp1-2; SALK_139194), STP4 (stp4-1; SALK_049432 and stp4-2; SALK_091229) and STP13 (stp13, SALK_0455494) loci. qPCR analyses revealed disruption of the STP1 gene expression in the stp1-1 mutant line (Fig EV1C), and a reduction of STP1 transcripts of 60% in the stp1-2 mutant (Fig EV1D). Furthermore, STP4 and STP13 transcript levels were reduced by approximately 40 and 80% in their respective mutant backgrounds compared to wild type (WT; Fig EV1C and D). To uncover putative functional relationship between the different STP isoforms, we generated the double mutant combinations stp1stp4 (from stp1-1 and stp4-1), stp1stp13 (from stp1-1), and stp4stp13 (from stp4-1). To describe morpho-physiological performance of the mutant lines in vivo, we used the automated phenotyping platform PlantScreen™ Compact System (PSI, Czech Republic). We established a robust phenotyping protocol to quantify daily, over a period of 8 days, plant morphological, physiological, and biochemical traits. Infrared thermography revealed that stp1stp4 plants had statistically significant higher leaf surface temperature compared to WT and all tested mutant combinations, even though the overall differences in surface temperatures were small (Fig 1A and B; Appendix Table S3). Given that leaf temperature is an indicator of stomatal aperture (Merlot et al, 2002), we hypothesized that stp1stp4 mutant plants may have closed stomata. Indeed, infrared gas analysis of stomatal conductance (gs) responses showed that light-induced stomatal opening was severely impaired in stp1stp4 plants (Fig 1C). Stomatal closure in response to darkness was also affected in this mutant (Fig 1C). The stp1-1 single mutant had a reduced steady-state gs. However, stp1-1 plants reached a similar overall gs amplitude as WT, but stomatal opening kinetics were slow (Fig 1C), well visible if gs values were normalized to values at the end of the night (EoN; Fig EV2A). The slow opening phenotype of stp1-1 single mutants was further confirmed in a second mutant allele stp1-2 (Fig EV2C and D). The mild stomatal opening phenotype of stp1 mutants can be explained by a strong upregulation of STP13 in the guard cells of mutant plants (Appendix Fig S1). STP13 might partially compensate for the loss of STP1 in the stp1 mutant. Interestingly, stp4-1 single mutants also had a reduced steady-state gs, but reached a greater overall gs amplitude compared to WT plants and showed similar stomatal opening kinetics (Figs 1C and EV2A). In addition, stp4-2 showed a similar elevated gs amplitude as the stp4-1 (Fig EV2C and D), indicating that mutation in the stp4 locus is responsible for the observed phenotype. Altogether, the phenotype of the single stp1-1 and stp4-1 mutants and their respective additional mutant alleles (stp1-2 and stp4-2), with gs amplitudes and stomatal opening kinetics similar to WT, suggests that STP1 and STP4 are both required to promote stomatal opening at the start of the day (Figs 1A–C and EV2A, C and D; Appendix Table S3). Despite the high expression of STP13 in guard cells (Fig EV1), the lack of functional STP13 in the stp13 single mutant did not cause a reduced gs amplitude nor slow opening kinetics. Stp13 mutants behaved similar to the stp4 mutant alleles (Figs 1A and B, and EV2E and F; Appendix Table S3). To investigate possible reasons behind the lack of phenotypes in stp4, stp13, stp1stp13, and stp4stp13, we performed STP gene expression analyses on guard cell-enriched epidermal peels of WT, stp4-1, and stp13 plants. Intriguingly, we found that STP1 was upregulated in guard cells of stp4-1 and stp13 plants (Appendix Fig S1), suggesting that STP1 might partially take over the role of STP4 and STP13 in their absence. In addition, STP13 is upregulated in response to pathogen infections or treatments with bacterial elicitors (Büttner, 2010), and it is the only STP gene inducible by osmotic stress, high salinity and abscisic acid (Yamada et al, 2011). STP13 function in guard cells may therefore become critical under stress conditions. Figure 1. Stomatal function is impaired in stp1stp4 plants Representative false color images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13, and stp4stp13 plants. Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point. Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1, and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype. Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype. Data information: (C, D) Plants have been illuminated with 150 μmol/m2/s white light after the end of the night (EoN) under ambient-air CO2 concentrations. (B) Asterisk (*) indicates significant statistical difference between WT and stp plants for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C, D) Different letters indicate significant statistical differences among genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Stomatal function in stp13 plants and genetic identity of WT-stp grafted plants Normalized whole-plant recordings of changes in stomatal conductance (gs-ginitial) from WT, stp1-1, stp4-1, and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype. Normalized whole-plant recordings of changes in stomatal conductance (gs-ginitial) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n ≥ 3 per genotype. Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-2 and stp4-2 plants. Data shown are means ± SEM; n = 4 per genotype. Normalized whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-2 and stp4-2 plants. Data shown are means ± SEM; n = 4 per genotype. Whole-plant recordings of changes in stomatal conductance (gs) from WT and stp13 plants. Data shown are means ± SEM; n = 3 per genotype. Normalized whole-plant recordings of changes in stomatal conductance (gs) from WT and stp13 plants. Data shown are means ± SEM; n = 3 per genotype. Representative molecular genotyping of roots from reciprocal grafted WT/stp1stp4 and stp1stp4/WT (shoot/root) plants. Roots from WT/WT and stp1stp4/stp1stp4 were used as a control. Genomic DNA extracted from roots was amplified using the genotyping primers listed in Appendix Table S2. For each grafted plant, PCR products were loaded according to the following order: STP1 gene-specific band, STP1 T-DNA-specific band, STP4 gene-specific band, STP4 T-DNA-specific band. Data information: (A - F) Plants have been illuminated with 150 μmol/m2/s white light after the end of the night (EoN) under ambient-air CO2 concentrations. (A, B, D, and F) gs values were normalized to values at the end of the night (EoN; 0 = ginitial). (C and E) Different letters indicate significant statistical differences among genotypes for the given time point for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (E and F) WT recordings are taken from Fig 1C. Download figure Download PowerPoint Previous studies reported the expression of STP1 and STP4 in Arabidopsis roots and their involvement in the uptake of monosaccharides from the rhizosphere (Truernit et al, 1996; Sherson et al, 2000; Yamada et al, 2011). To rule out the possibility that simultaneous knock-out of STP1 and STP4 in the roots contributed to the severe impairment of stomatal opening in stp1stp4, we measured gs in WT/stp1stp4 and stp1stp4/WT grafted plants. Plants with WT shoots and stp1stp4 roots showed stomatal conductance comparable to WT, whereas plants bearing stp1stp4 shoots and WT roots displayed a stp1stp4-like phenotype (Figs 1D and EV2B). The genetic identity of the roots from the reciprocal grafted plants was confirmed by molecular genotyping (Fig EV2G). Our grafting experiments indicate that the stomatal phenotype of stp1stp4 is independent from the function of these transporters in the roots and further support their essential role in guard cells. stp1stp4 guard cells have reduced levels of glucose We reasoned that inhibition of light-induced stomatal opening in stp1stp4 mutants might be a direct consequence of impaired Glc and/or Fru import to guard cells. To test this hypothesis, we measured soluble sugar content in guard cells of intact leaves of WT and stp1stp4 mutant plants at the EoN and after 40 min of light (Fig 2A). In WT guard cells, the levels of Glc and Suc were unaltered in response to the light treatment, while Fru levels decreased to half due to illumination (Fig 2A). The sustained levels of Suc are likely due to continuous Suc import from the mesophyll, which is consistent with the high expression of SUC1 and SUC3 transporters in guard cells at the EoN (Fig EV3A). Notably, stp1stp4 guard cells had significantly lower amounts of Glc and Fru at the EoN compared to WT, and Glc levels were almost undetectable after 40 min of light (Fig 2A). Suc surprisingly accumulated to higher levels (Fig 2A), perhaps as a result of reduced consumption, sequestration in the vacuole or SUC transporters upregulation to compensate for the loss of STPs. In contrast to stp1stp4 double mutants, guard cells of single stp1-1 and stp4-1 mutants contained similar amounts of sugars as that of WT guard cells (Fig EV3B). However, there was a trend in stp1-1 guard cells towards reduced Glc levels compared to WT (Fig EV3B), which is in line with the mild impairment in stomatal opening in these plants. Figure 2. stp1stp4 plants have low levels of hexoses and are devoid of starch in guard cells Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 μmol/m2/s following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point. Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1, and suc3 plants. Scale bar, 10 μm. Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1, and suc3 plants at the end of the night (EoN) and after 1 and 3 h of illumination with 150 μmol/m2/s of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point. Data information: (A) Different letters indicate significant statistical differences among time points for the given genotype. Asterisk (*) indicates significant statistical difference between genotypes for the given time point for P< 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C) Different letters indicate significant statistical differences among genotypes for the given time point. Asterisk (*) indicates significant statistical difference among time points for the given genotypes for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Guard cell metabolites and gene expression in stp mutants SUC1 and SUC3 gene transcript levels in WT guard cell-enriched epidermal peels compared to WT rosette leaves at the end of the night. KAT1 and MYB60 were used as guard cell-specific markers, whereas BAM3 was used as leaf-specific marker. Data for two independent experiments are shown; means ± fold change range n ≥ 5. Content of soluble sugars in guard cell-enriched epidermal peels of WT, stp1-1, and stp4-1 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 μmol/m2/s following the EoN. Data shown are means ± SEM; n = 3 for WT and n ≥ 5 for the mutants per time point. Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp13 plants. Scale bar, 10 μm. Starch dynamics in guard cells of intact leaves of WT and stp13 plants at the end of the night (EoN) and after 1 and 3 h of illumination with 150 μmol/m2/s of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point. Data information: (A) ACT2 was used as a housekeeping gene for normalization. For details about fold change and error calculations, see Materials and Methods section. Primer sequences and efficiencies are given in Appendix Table S2. (B, D) Different letters indicate significant statistical differences among genotypes for the given time point. Asterisk (*) indicates significant statistical differences among time points for the given genotype for P < 0.05 determined by one-way ANOVA with post hoc Tukey's test. (C, D) WT data are taken from Fig 2B and C. Download figure Download PowerPoint These data demonstrate in vivo the function of STP1 and STP4 in the coordinate transport of Glc and to a lesser extent Fru at the guard cell plasma membrane during light-induced stomatal opening and likely explain the inability of stp1stp4 to open stomata in the light. Based on the phenotype of the stp1stp4 mutant, which cannot open stomata despite the high levels of Suc, we suggest that imported Glc provides a major source of carbon for light-induced stomatal opening at the start of the day. stp1stp4 guard cells are devoid of starch Given that guard cells possess several characteristics of sink tissues with fewer and smaller chloroplasts and low photosynthetic rates, we next investigated whether the import of apoplastic Glc by STPs influenced guard cell starch metabolism. As we reported previously (Horrer et al, 2016), starch was degraded in WT guard cells when the plants were illuminated, coinciding with the opening of the stomata (Fig 2B and C); after falling to near zero in the first hour after dawn, starch levels then began to rise again (Fig 2B and C). Notably, at the EoN, stp1stp4 guard cells were essentially devoid of starch, and no starch synthesis occurred during the first 3 h of light (Fig 2B and C). The single stp mutants displayed a milder phenotype. Although they degraded the starch in guard cells upon transition to light similar to WT, they failed to resynthesize it (Figs 2B and C, and EV3C and D). The fact that stp1stp4 guard cells were unable to make starch even in the presence of high amounts of Suc led us to hypothesize that at the start of the day mesophyll-derived Glc imported by STPs is the precursor for guard cell starch biosynthesis. If this hypothesis is correct, Arabidopsis plants lacking SUC transporters should be able to make starch under the investigated conditions. Indeed, as we show in Fig 2B and C, guard cells of suc1 and suc3 mutants had essentially normal starch turnover during the first 3 h of light (Fig 2B and C). stp1stp4 stomatal phenotype impacts on plant photosynthesis and growth Nearly all CO2 needed for mesophyll photosynthesis enters the plant waxy leaf epidermis through the stomatal pores. Consequently, we predicted that reduced stomatal aperture in stp1stp4 should have a major impact on intercellular CO2 concentrations (Ci, μmol CO2 mol/ai