Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function

Water is a principal limitation to agricultural production during drought and in arid regions of the world. Mechanisms that plants use to cope with drought can be grouped into two different strategies: drought tolerance and drought avoidance. Previous efforts toward engineering plants for improved performance during drought have focused on drought tolerance, the ability to adjust to dry conditions. This report addresses the engineering of a drought-avoidance phenotype, which allows for the conservation of water during plant growth. The majority of water lost from plants occurs through stomata. When stomata are open, potassium, chloride and/or malate are present at high concentrations in guard cells. The accumulation of large numbers of ions during stomatal opening increases the turgor pressure of the guard cells, which results in increased pore size. Expression of a single gene from maize, NADP-malic enzyme (ME), which converts malate and NADP to pyruvate, NADPH, and CO(2), resulted in altered stomatal behaviour and water relations in tobacco. The ME-transformed plants had decreased stomatal conductance and gained more fresh mass per unit water consumed than did the wild type, but they were similar to the wild type in their growth and rate of development. Providing chloride via the transpiration stream partially reversed the effects of ME expression on stomatal aperture size, which is consistent with the interpretation that expression of ME altered malate metabolism in guard cells. These results suggest a role for malic enzyme in the mechanism of stomatal closure, as well as a potential mechanism for genetically altering plant water use.     

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

The barley anion channel,HvALMT1, has multiple roles in guard cell physiology and grain metabolism

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue‐specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18–30% of wild‐type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue‐specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed.     

Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.     

Alterations in light-induced stomatal opening in a starch-deficient mutant of Arabidopsis thaliana L. deficient in chloroplast phosphoglucomutase activity

Stomatal responses to light of Arabidopsis thaliana wild-type plants and mutant plants deficient in starch (phosphoglucomutase deficient) were compared in gas exchange experiments. Stomatal density, size and ultrastructure were identical for the two phenotypes, but no starch was observed in guard cells of the mutant plants whatever the time of day. The overall extent of changes in stomatal conductance during 14 h light–10 h dark cycles was similar for the two phenotypes. However, the slow endogenous stomatal opening occurring in darkness in the wild type was not observed in the mutant plants. Stomata in the mutant plants responded much more slowly to blue light (70 μmol m^?2 s^?1) though the response to red light (250 μmol m^?2 s^?1) was similar to that of wild-type plants. In paradermal sections, stomatal responses to red light (300 μmol m^?2 s^?1) were weak for wild-type plants as well as for mutant plants. Stomatal opening was greater under low blue light (75 μmol m^?2 s^?1) than under red light for the two genotypes. However, in mutant plants, a high chloride concentration (50 mol m^?3) was necessary to achieve the same stomatal aperture as observed for the wild-type plants. These results suggest that starch metabolism, via the synthesis of a counter-ion to potassium (probably malate), is required for full stomatal response to blue light but is not involved in the stomatal response to red light.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H₂O₂ and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H₂O₂ levels as the wild type in response to ABA. However, ABA- or H₂O₂-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H₂O₂ in ABA-induced stomatal closure.     

Environmental regulation of stomatal response in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO₂] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO₂]-induced and low humidity-induced stomatal closure.     

Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution

Stomatal pores evolved more than 410 million years ago [1,?2]?and allowed vascular plants to regulate transpirational water loss during the uptake of CO(2) for photosynthesis [3]. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens [2] respond to environmental signals in a similar way to those of flowering plants [4] and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response [5] is?involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) [6], is rescued by substitution with the moss P.?patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P.?patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago [7], prior to the evolution of ferns [8, 9].     

The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana

Gas exchange parameters and stomatal physical properties were measured in Tradescantia virginiana plants grown under well-watered conditions and treated daily with either distilled water (control) or 3.0 mM abscisic acid (ABA). Photosynthetic capacity (CO(2) assimilation rate for any given leaf intercellular CO(2) concentration [c(i)]) and relative stomatal sensitivity to leaf-to-air vapor-pressure difference were unaffected by the ABA treatment. However, at an ambient CO(2) concentration (c(a)) of 350 micromol mol(-1), ABA-treated plants operated with significantly lower c(i). ABA-treated plants had significantly smaller stomata and higher stomatal density in their lower epidermis. Stomatal aperture versus guard cell pressure (P(g)) characteristics measured with a cell pressure probe showed that although the form of the relationship was similar in control and ABA-treated plants, stomata of ABA-treated plants exhibited more complete closure at P(g) = 0 MPa and less than half the aperture of stomata in control plants at any given P(g). Scaling from stomatal aperture versus P(g) to stomatal conductance versus P(g) showed that plants grown under ABA treatment would have had significantly lower maximum stomatal conductance and would have operated with lower stomatal conductance for any given guard cell turgor. This is consistent with the observation of lower c(i)/c(a) in ABA-treated plants with a c(a) of 350 micromol mol(-1). It is proposed that the ABA-induced changes in stomatal mechanics and stomatal conductance versus P(g) characteristics constitute an improvement in water-use efficiency that may be invoked under prolonged drought conditions.     

The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development

Wild-type stomata are distributed nonrandomly, and their density is controlled by endogenous and exogenous factors. In the Arabidopsis mutant stomatal density and distribution1-1 (sdd1-1), the establishment of the stomatal pattern is disrupted, resulting in stomata clustering and twofold to fourfold increases in stomatal density. The SDD1 gene that encodes a subtilisin-like Ser protease is expressed strongly in stomatal precursor cells (meristemoids and guard mother cells), and the SDD1 promoter is controlled negatively by a feedback mechanism. The encoded protein is exported to the apoplast and probably is associated with the plasma membrane. SDD1 overexpression in the wild type leads to a phenotype opposite to that caused by the sdd1-1 mutation, with a twofold to threefold decrease in stomatal density and the formation of arrested stomata. While SDD1 overexpression was effective in the flp mutant, the tmm mutation acted epistatically. Thus, we propose that SDD1 generates an extracellular signal by meristemoids/guard mother cells and demonstrate that the function of SDD1 is dependent on TMM activity.     

Regulation of Dark-Induced Stomatal Closure in <Emphasis Type="Italic">Arabidopsis</Emphasis> Dynamin-Like Protein 1E (<Emphasis Type="Italic">adl1e</Emphasis>) Mutant Leaves

Arabidopsis thaliana dynamin-like protein 1E (ADL1E) is known to regulate mitochondrial elongation. The adl1e mutant has no morphological phenotype, and the growth and photosynthetic activity of the mutant are similar to those of the wild type. Leaf O₂ uptake, which is supported by mitochondrial activity in the dark, is increased 1.7-fold by mutation in adl1e gene. The ATP content in the dark of guard and mesophyll cell protoplasts (GCPs and MCPs, respectively) was 2.5- to 4-fold higher in GCPs of the mutant and the wild type, and increased upon the addition of glucose in both genotypes. Oligomycin, an inhibitor of mitochondrial ATPase, suppressed ATP synthesis in both GCPs and MCPS isolated from adl1e plants, indicating that mutant had higher mitochondrial activity. The stomatal apertures of mutant and wild-type plants were then analyzed in vitro. In the light, the stomata of both genotypes showed similar patterns of opening. However, in the dark response, the stomata of the adl1e mutant closed faster than did those of the wild type. Oligomycin severely inhibited dark-induced stomatal closure in both cell types. The results suggest that stomatal closure in the dark is governed by cytosolic ATP concentration, which is stimulated by mitochondrial activity.     

Differential requirement for NO during ABA-induced stomatal closure in turgid and wilted leaves

Abscisic acid (ABA)-induced stomatal closure is mediated by a complex, guard cell signalling network involving nitric oxide (NO) as a key intermediate. However, there is a lack of information concerning the role of NO in the ABA-enhanced stomatal closure seen in dehydrated plants. The data herein demonstrate that, while nitrate reductase (NR)1-mediated NO generation is required for the ABA-induced closure of stomata in turgid leaves, it is not required for ABA-enhanced stomatal closure under conditions leading to rapid dehydration. The results also show that NO signalling in the guard cells of turgid leaves requires the ABA-signalling pathway to be both capable of function and active. The alignment of this NO signalling with guard cell Ca²⁺-dependent/independent ABA signalling is discussed. The data also highlight a physiological role for NO signalling in turgid leaves and show that stomatal closure during the light-to-dark transition requires NR1-mediated NO generation and signalling.     

Hexokinase mediates stomatal closure

Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO₂ for photosynthesis with the loss of water through transpiration. Increased guard‐cell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar‐phosphorylating enzyme involved in sugar‐sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback‐inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.     

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2, abscisic acid,darkness, vapor pressure deficit and ozone

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂ induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂ and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).     

Stomatal response characteristics of Tradescantia virginiana grown at high relative air humidity

Plants produced at high relative air humidity (RH) show poor control of water loss after transferring to low RH, a phenomenon which is thought to be due to their stomatal behaviour. The stomatal anatomy and responses of moderate (55%) and high (90%) RH grown Tradescantia virginiana plants to treatments that normally induce stomatal closure, i.e. desiccation, abscisic acid (ABA) application and exposure to darkness were studied using attached or detached young, fully expanded leaves. Compared with plants grown at moderate RH the transpiration rate, stomatal conductance and aperture of high RH grown plants measured at the same condition (40% RH) were, respectively, 112, 139 and 132% in light and 141, 188 and 370% in darkness. Besides the differences in stomatal size (guard cell length was 56.7 and 73.3 µm for moderate and high RH grown plants, respectively), there was a clear difference in stomatal behaviour. The stomata responded to desiccation, ABA and darkness in both moderate and high RH grown plants, but the high variability of stomatal closure in high RH grown plants was striking. Some stomata developed at high RH closed in response to darkness or to a decrease in relative water content to the same extent as did stomata from moderate RH grown plants, whereas others closed only partly or did not close at all. Evidently, some as yet unidentified physiological or anatomical changes during development disrupt the normal functioning of some stomata in leaves grown at high RH. The failure of some stomata to close fully in response to ABA suggests that ABA deficiency was not responsible for the lack of stomatal closure in response to desiccation.