H2S is involved in drought-mediated stomatal closure through PLDα1 in Arabidopsis.

MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.

Simultaneous and Independent Abaxial and Adaxial Gas Exchange Measurements.

Stomata can be distributed exclusively on the abaxial or adaxial leaf surface, but they are most commonly found on both leaf surfaces. Variations in stomatal arrangement, patterning, and the impact on photosynthesis can be measured using an infrared gas exchange system. However, when using standard gas exchange techniques, both surfaces are measured together and averaged to provide leaf-level values. Employing an innovative gas exchange apparatus with two infrared gas analyzers, separate gaseous flux from both leaf surfaces can be quantified simultaneously and independently. Here, we provide examples of typical measurements that can be performed using a "split chamber" gas exchange system.

Effect of tropospheric ozone and its protectants on gas exchange parameters, antioxidant enzymes and quality of Garlic (Allium sativum. L).

An experimental study was conducted to assess the detrimental effect of ground-level ozone (O3) on garlic physiology and to find out appropriate control measures against ground-level O3, at TNAU-Horticultural Research farm, Udhagamandalam. Elevated ground ozone levels significantly decreased garlic leaf chlorophyll, photosynthetic rate, stomatal conductance, total soluble solids and pungency. The garlic chlorophyll content was highest in ambient ozone level and lowest in elevated ozone@200 ppb, highest stomatal conductance was recorded in ambient ozone with foliar spray of 3%Panchagavya, and the lowest was observed in elevated ozone@200 ppb. Since the elevated O3 had reduced in garlic photosynthetic rate significantly the lowest was observed in elevated O3@200 ppb and the highest photosynthetic rate was observed in ambient Ozone with foliar spray 3% of panchagavya after a week. The antioxidant enzymes of garlic were increased with increased concentration of tropospheric ozone. The highest catalase (60.97 µg of H2O2/g of leaf) and peroxidase (9.13 ΔA/min/g of leaf) concentration was observed at 200 ppb elevated ozone level. Garlic pungency content was highest in ambient ozone with foliar spray of 0.1% ascorbic acid and the lowest was observed under elevated O3@200 ppb. Highest total soluble solids were observed in ambient ozone with foliar spray of 3%Panchagavya and the lowest observed in elevated ozone@200 ppb. Thus, tropospheric ozone has a detrimental impact on the physiology of crops, which reduced crop growth and yield. Under elevated O3 levels, ascorbic acid performed well followed by panchagavya and neem oil. The antioxidant such as catalase and peroxidase had positive correlation among themselves and had negative correlation with chlorophyll content, stomatal conductance, photosynthetic rate, pungency and TSS. The photosynthetic rate has high positive correlation with chlorophyll content, pungency and TSS. Correlation analysis confirmed the negative effects of tropospheric ozone and garlic gas exchange parameters and clove quality. The ozone protectants will reduce stomatal opening by which the entry of O3 in to the cell will be restricted and other hand they also will alleviate ROS and allied stresses.

MYC2 regulates stomatal density and water use efficiency via targeting EPF2/EPFL4/EPFL9 in poplar.

Although polyploid plants have lower stomatal density than their diploid counterparts, the molecular mechanisms underlying this difference remain elusive. Here, we constructed a network based on the triploid poplar transcriptome data and triple-gene mutual interaction algorithm and found that PpnMYC2 was related to stomatal development-related genes PpnEPF2, PpnEPFL4, and PpnEPFL9. The interactions between PpnMYC2 and PagJAZs were experimentally validated. PpnMYC2-overexpressing poplar and Arabidopsis thaliana had reduced stomatal density. Poplar overexpressing PpnMYC2 had higher water use efficiency and drought resistance. RNA-sequencing data of poplars overexpressing PpnMYC2 showed that PpnMYC2 promotes the expression of stomatal density inhibitors PagEPF2 and PagEPFL4 and inhibits the expression of the stomatal density-positive regulator PagEPFL9. Yeast one-hybrid system, electrophoretic mobility shift assay, ChIP-qPCR, and dual-luciferase assay were employed to substantiate that PpnMYC2 directly regulated PagEPF2, PagEPFL4, and PagEPFL9. PpnMYC2, PpnEPF2, and PpnEPFL4 were significantly upregulated, whereas PpnEPFL9 was downregulated during stomatal formation in triploid poplar. Our results are of great significance for revealing the regulation mechanism of plant stomatal occurrence and polyploid stomatal density, as well as reducing stomatal density and improving plant water use efficiency by overexpressing MYC2.

Ethylene inhibits ABA-induced stomatal closure via regulating NtMYB184-mediated flavonol biosynthesis in tobacco.

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.

OnGuard3e: A predictive, ecophysiology-ready tool for gas exchange and photosynthesis research.

Gas exchange across the stomatal pores of leaves is a focal point in studies of plant-environmental relations. Stomata regulate atmospheric exchange with the inner air spaces of the leaf. They open to allow CO2 entry for photosynthesis and close to minimize water loss. Models that focus on the phenomenology of stomatal conductance generally omit the mechanics of the guard cells that regulate the pore aperture. The OnGuard platform fills this gap and offers a truly mechanistic approach with which to analyse stomatal gas exchange, whole-plant carbon assimilation and water-use efficiency. Previously, OnGuard required specialist knowledge of membrane transport, signalling and metabolism. Here we introduce OnGuard3e, a software package accessible to ecophysiologists and membrane biologists alike. We provide a brief guide to its use and illustrate how the package can be applied to explore and analyse stomatal conductance, assimilation and water use efficiencies, addressing a range of experimental questions with truly predictive outputs.

Growth, physiology, and stomatal parameters of plant polyploids grown under ice age, present-day, and future CO2 concentrations.

Polyploidy plays an important role in plant evolution, but knowledge of its eco-physiological consequences, such as of the putatively enlarged stomata of polyploid plants, remains limited. Enlarged stomata should disadvantage polyploids at low CO2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO2 uptake. We observed the growth, physiology, and epidermal cell features of 15 diploids and their polyploid relatives cultivated under glacial, present-day, and potential future atmospheric CO2 concentrations (200, 400, and 800 ppm respectively). We demonstrated some well-known polyploidy effects, such as faster growth and larger leaves, seeds, stomata, and other epidermal cells. The stomata of polyploids, however, tended to be more elongated than those of diploids, and contrary to common belief, they had no negative effect on the CO2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO2 concentrations. Higher polyploids with large genomes also showed increased operational stomatal conductance and consequently, a lower water-use efficiency. Our results point to a possible decrease in growth superiority of polyploids over diploids in a current and future high CO2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

Phytomelatonin interferes with flavonols biosynthesis to regulate ROS production and stomatal closure in tobacco.

Flavonols are well-known antioxidants that prevent stomatal closure via interfering with ROS signaling. Phytomelatonin regulates stomatal closure, but the signaling pathways are still largely unknown. Here, we investigated the role of flavonols in phytomelatonin-mediated stomatal closure in tobacco plants. The application of melatonin induced stomatal closure through NADPH oxidase-mediated ROS production. Transgenic tobacco plants overexpressing soybean GmSNAT1 (coding for serotonin N-acetyltransferase that catalyzes the penultimate step in phytomelatonin biosynthesis) had higher phytomelatonin concentration, accumulated more ROS in guard cells and were more sensitive to melatonin-induced stomatal closure than the wild-type plants, which was associated with the higher expression of PMTR1-homologous genes. Exogenous melatonin decreased flavonol concentrations in guard cells and the expression of flavonoid-related genes in wild-type and transgenic tobacco plants, and these inhibitory effects were more obvious in GmSNAT1-overexpressing plants than the wild type. However, the melatonin-mediated stomatal closure and ROS production were diminished by the application of kaempferol (a type of flavonol). Additionally, transgenic tobacco plants with increased expression of NtFLS (encoding flavonol synthase) were less sensitive to melatonin-induced stomatal closure. In conclusion, phytomelatonin hampers the biosynthesis of flavonols in guard cells, which results in high concentration of ROS and induces stomatal closure in tobacco plants.

Changes in endogenous abscisic acid and stomata of the resurrection fern, Pleopeltis polypodioides, in response to de- and rehydration.

PREMISE: While angiosperms respond uniformly to abscisic acid (ABA) by stomatal closure, the response of ferns to ABA is ambiguous. We evaluated the effect of endogenous ABA, hydrogen peroxide (H2 O2 ), nitric oxide (NO), and Ca2+ , low and high light intensities, and blue light (BL) on stomatal opening of Pleopeltis polypodioides. METHODS: Endogenous ABA was quantified using gas chromatography-mass spectrometry; microscopy results and stomatal responses to light and chemical treatments were analyzed with Image J. RESULTS: The ABA content increases during initial dehydration, peaks at 15 h and then decreases to one fourth of the ABA content of hydrated fronds. Following rehydration, ABA content increases within 24 h to the level of hydrated tissue. The stomatal aperture opens under BL and remains open even in the presence of ABA. Closure was strongly affected by BL, NO, and Ca2+ , regardless of ABA, H2 O2 effect was weak. CONCLUSIONS: The decrease in the ABA content during extended dehydration and insensitivity of the stomata to ABA suggests that the drought tolerance mechanism of Pleopeltis polypodioides is independent of ABA.

A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

Light, rather than circadian rhythm, regulates gas exchange in ferns and lycophytes.

Circadian regulation plays a vital role in optimizing plant responses to the environment. However, while circadian regulation has been extensively studied in angiosperms, very little is known for lycophytes and ferns, leaving a gap in our understanding of the evolution of circadian rhythms across the plant kingdom. Here, we investigated circadian regulation in gas exchange through stomatal conductance and photosynthetic efficiency in a phylogenetically broad panel of 21 species of lycophytes and ferns over a 46 h period under constant light and a selected few under more natural conditions with day-night cycles. No rhythm was detected under constant light for either lycophytes or ferns, except for two semi-aquatic species of the family Marsileaceae (Marsilea azorica and Regnellidium diphyllum), which showed rhythms in stomatal conductance. Furthermore, these results indicated the presence of a light-driven stomatal control for ferns and lycophytes, with a possible passive fine-tuning through leaf water status adjustments. These findings support previous evidence for the fundamentally different regulation of gas exchange in lycophytes and ferns compared to angiosperms, and they suggest the presence of alternative stomatal regulations in Marsileaceae, an aquatic family already well known for numerous other distinctive physiological traits. Overall, our study provides evidence for heterogeneous circadian regulation across plant lineages, highlighting the importance of broad taxonomic scope in comparative plant physiology studies.

Transcriptome and anatomical studies reveal alterations in leaf thickness under long-term drought stress in tobacco.

Drought is one of the foremost environmental factors that limit the growth of plants. Leaf thickness (LT) is an important quantitative trait in plant physiology. The experiment was carried out in a growth room and the plants were divided into two groups such as well-watered and drought-stressed. This work investigated leaf growth in terms of leaf surface growth and expansion rate, leaf stomata traits, LT, anticlinal growth, and leaf cell layers. The results showed that the leaf area and leaf surface expansion rate were decreased by drought stress (DS). Similarly, LT, anticlinal expansion rate, palisade and spongy tissue thickness, and their related expansion rates were also decreased at different days' time points (DTP) of DS. However, a steady increase was observed in the aforementioned parameters after 12 DTP of DS. The stomatal density increased while stomata size decreased at 3 DTP and 12 DTP (low leaf water potential and relative leaf water content at these time points) and vice versa at 24 DTP compared with the well-watered plants indicating adaptations in these traits in response to DS, and thus the leaf water status played a role in the regulation of leaf stomata traits. The cell length decreased in the upper epidermis, palisade and spongy tissues by DS up to 12 DTP led to lower LT while an increase was observed after 12 DTP that resulted in higher LT. The increase in the LT was supported by the upregulation of starch and sucrose metabolism, glycerolipid metabolism, protein processing in endoplasmic reticulum pathways at 18 DTP along with the differentially expressed genes induced that were related to cell wall remodeling (cellulose, expansin, xyloglucans) and cell expansion (auxin response factors and aquaporin). The results explain the response of leaf thickness to drought stress and show alterations in LT and leaf stomatal traits. This study might serve as a valuable source of gene information for functional studies and provide a theoretical basis to understand leaf growth in terms of leaf anatomy and leaf stomatal traits under drought stress.

Saussurea involucrata PIP2;4 improves growth and drought tolerance in Nicotiana tabacum by increasing stomatal density and sensitivity.

Aquaporins, the major facilitators of water transport across membranes, are involved in growth and development and adaptation to drought stress in plants. In this study, a plasma membrane intrinsic protein (SiPIP2;4) was cloned from Saussurea involucrata, a cold-tolerant hardy herb. The expression of SiPIP2;4 increased the stomatal density and sensitivity of tobacco (Nicotiana tabacum), thus, affecting the plant's growth and resistance to the diverse water environment. The higher stomatal density under well-watered conditions effectively promoted the photosynthetic rate, which led to the rapid growth of transgenic lines. The stomata in the transgenic lines responded more sensitively to the vapor pressure deficit than the wild-type under different levels of ambient humidity. Their stomatal apertures positively correlated with the ambient humidity. Under drought conditions, the overexpression of SiPIP2;4 promoted rapid stomatal closure, reduced water dissipation, and enhanced drought tolerance. These results indicate that SiPIP2;4 regulates the density and sensitivity of plant stomata, thus, playing an important role in balancing plant growth and stress tolerance. This suggests that SiPIP2;4 has the potential to serve as a genetic resource for crop improvement.

Elevated CO2 induces rapid dephosphorylation of plasma membrane H+ -ATPase in guard cells.

Light induces stomatal opening, which is driven by plasma membrane (PM) H+ -ATPase in guard cells. The activation of guard-cell PM H+ -ATPase is mediated by phosphorylation of the penultimate C-terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard-cell PM H+ -ATPase in Arabidopsis thaliana. Immunodetection of guard-cell PM H+ -ATPase, time-resolved leaf gas-exchange analyses and stomatal aperture measurements were carried out. We found that light-dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post-transition. Gas-exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard-cell PM H+ -ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse-genetic analyses using guard-cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light-dark transition. Our results indicate that CO2 -mediated dephosphorylation of guard-cell PM H+ -ATPase underlies stomatal closure.

Reactive Carbonyl Species Inhibit Blue-Light-Dependent Activation of the Plasma Membrane H+-ATPase and Stomatal Opening.

Reactive oxygen species (ROS) play a central role in plant responses to biotic and abiotic stresses. ROS stimulate stomatal closure by inhibiting blue light (BL)-dependent stomatal opening under diverse stresses in the daytime. However, the stomatal opening inhibition mechanism by ROS remains unclear. In this study, we aimed to examine the impact of reactive carbonyl species (RCS), lipid peroxidation products generated by ROS, on BL signaling in guard cells. Application of RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), inhibited BL-dependent stomatal opening in the epidermis of Arabidopsis thaliana. Acrolein also inhibited H+ pumping and the plasma membrane H+-ATPase phosphorylation in response to BL. However, acrolein did not inhibit BL-dependent autophosphorylation of phototropins and the phosphorylation of BLUE LIGHT SIGNALING1 (BLUS1). Similarly, acrolein affected neither the kinase activity of BLUS1 nor the phosphatase activity of protein phosphatase 1, a positive regulator of BL signaling. However, acrolein inhibited fusicoccin-dependent phosphorylation of H+-ATPase and stomatal opening. Furthermore, carnosine, an RCS scavenger, partially alleviated the abscisic-acid- and hydrogen-peroxide-induced inhibition of BL-dependent stomatal opening. Altogether, these findings suggest that RCS inhibit BL signaling, especially H+-ATPase activation, and play a key role in the crosstalk between BL and ROS signaling pathways in guard cells.

Plant signaling: Peptide-receptor pair re-opens stomata after pathogen infection.

Stomata - cellular valves in the epidermis of land plants - close their apertures to prevent water loss or pathogen entry. A new study now reports that the plant immune response induces the expression of a peptide ligand-receptor pair that re-opens stomata to resume gas exchange and transpiration after pathogen infection.

Functional roles of ALMT-type anion channels in malate-induced stomatal closure in tomato and Arabidopsis.

Guard-cell-type aluminium-activated malate transporters (ALMTs) are involved in stomatal closure by exporting anions from guard cells. However, their physiological and electrophysiological functions are yet to be explored. Here, we analysed the physiological and electrophysiological properties of the ALMT channels in Arabidopsis and tomato (Solanum lycopersicum). SlALMT11 was specifically expressed in tomato guard cells. External malate-induced stomatal closure was impaired in ALMT-suppressed lines of tomato and Arabidopsis, although abscisic acid did not influence the stomatal response in SlALMT11-knock-down tomato lines. Electrophysiological analyses in Xenopus oocytes showed that SlALMT11 and AtALMT12/QUAC1 exhibited characteristic bell-shaped current-voltage patterns dependent on extracellular malate, fumarate, and citrate. Both ALMTs could transport malate, fumarate, and succinate, but not citrate, suggesting that the guard-cell-type ALMTs are dicarboxylic anion channels activated by extracellular organic acids. The truncation of acidic amino acids, Asp or Glu, from the C-terminal end of SlALMT11 or AtALMT12/QUAC1 led to the disappearance of the bell-shaped current-voltage patterns. Our findings establish that malate-activated stomatal closure is mediated by guard-cell-type ALMT channels that require an acidic amino acid in the C-terminus as a candidate voltage sensor in both tomato and Arabidopsis.

Stomata on the abaxial and adaxial leaf surfaces contribute differently to leaf gas exchange and photosynthesis in wheat.

Although stomata are typically found in greater numbers on the abaxial surface, wheat flag leaves have greater densities on the adaxial surface. We determine the impact of this less common stomatal patterning on gaseous fluxes using a novel chamber that simultaneously measures both leaf surfaces. Using a combination of differential illuminations and CO2 concentrations at each leaf surface, we found that mesophyll cells associated with the adaxial leaf surface have a higher photosynthetic capacity than those associated with the abaxial leaf surface, which is supported by an increased stomatal conductance (driven by differences in stomatal density). When vertical gas flux at the abaxial leaf surface was blocked, no compensation by adaxial stomata was observed, suggesting each surface operates independently. Similar stomatal kinetics suggested some co-ordination between the two surfaces, but factors other than light intensity played a role in these responses. Higher photosynthetic capacity on the adaxial surface facilitates greater carbon assimilation, along with higher adaxial stomatal conductance, which would also support greater evaporative leaf cooling to maintain optimal leaf temperatures for photosynthesis. Furthermore, abaxial gas exchange contributed c. 50% to leaf photosynthesis and therefore represents an important contributor to overall leaf gas exchange.

Gas Exchange Measurements in Systemic Signaling Studies.

Different parts of a plant can be simultaneously exposed to very different conditions, for example a leaf moving in and out of shadow. In addition to local responses, transmission of information between different tissues and organs is thought to affect the coordination of overall responses to changing environmental conditions. An important adaptive role is played by the stomata, which regulate the evaporation of water vapor and supply of CO2 for photosynthesis. Here, we describe a method to study the effect of distally triggered systemic signals on stomatal conductance. The experimental set up, consisting of a growth chamber and a leaf gas exchange measuring system, enables time-resolved measurements on an intact leaf while maintaining a full control over the environmental conditions of the measured leaf and the whole seedling. The method can be used as a powerful tool to study short- and long-term stomatal responses to changes in different environmental variables, such as light.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.