Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membra... more Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and Q...
Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked b... more Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone (O3 ). Such O3 -pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O3 pulses (200 to 1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic Ca2+ level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.
Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasio... more Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to which 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 on JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2, and npr1 alleles. While stomata in the studied mutants clearly responded to ABA, CO2 , light, and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated comparing with the wild type. Stomatal re-opening 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 the treatments, 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 h and reached a maximum in 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 whole-plant stomatal response to environmental cues in Arabidopsis and tomato.
Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress... more Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 s...
Stomata, small pores on the surfaces of leaves formed by a pair of guard cells, adapt rapidly to ... more Stomata, small pores on the surfaces of leaves formed by a pair of guard cells, adapt rapidly to changes in the environment by adjusting the aperture width. As a long term response, the number of stomata is regulated during stomatal development. The hormone abscisic acid (ABA) regulates both processes. In ABA mediated guard cell signaling the protein kinase OPEN STOMATA1 (OST1) has a central role, as stomatal closure in the ost1 mutant is impaired in response to ABA and to different environmental stimuli. We aimed to dissect the contribution of different ABA-related regulatory mechanisms in determining stomatal conductance, a combination of stomatal density and aperture width, and crossed the ost1 mutant with mutants that either decreased (aba3) or increased (cyp707a1/a3) the concentration of ABA in plants. The double mutant ost1 aba3 had higher stomatal conductance than either parent due to a combination of increased stomatal aperture width and higher stomatal density. In the tripl...
Abstract Optimization between photosynthetic carbon assimilation and stomatal water loss is the k... more Abstract Optimization between photosynthetic carbon assimilation and stomatal water loss is the key in order to breed crops for future climate. We analyzed stomatal conductance (gs), CO2 assimilation rate (Anet), fungal diseases, grain yield and yield components of seven European malting barley genotypes treated with fungicides alone or together with biostimulants in the field over three consecutive seasons. Stomatal conductance, net assimilation rate and grain yield were affected by genotype, treatment and year. We then examined which of these traits are most strongly correlated with yield. Grain yields ranged between 6000-8300 kg ha−1 in 2015 and 2017, but only 3500–6000 kg ha−1 in 2016 due to hotter and drier weather. The benefits of treatments with fungicides or fungicides together with biostimulants were greater in 2016, when the treatments increased yields by 20–21% on average, compared to 0–11% in 2015 and 2017. In 2016, gs and Anet were correlated with grain yield, indicating that in hotter and drier than average season maintaining higher transpiration and photosynthesis resulted in higher yield. In 2015 and 2017, average values of instantaneous gs and Anet were not significantly correlated with grain yield. Pooling all years, longer pre-heading period, higher grain numbers per ha and per ear, larger 1000-kernel weight and higher water use efficiency were associated with higher grain yield, whereas a negative correlation was detected between grain yield and the number of ears per area. Thus, developing fewer productive tillers per area, but more and heavier grains per ear led to higher grain yield. Major barley fungal pathogens had a negative effect on grain yield via shortening of grain filling period.
Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptak... more Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion current...
Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. C... more Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. CO2 elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO2/ABA interaction remain unclear. Two models have been considered: (i) CO2 elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (ii) CO2 signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutants nced3/nced5 and aba2-1 remain responsive to CO2 elevation. Rapid CO2-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays ...
The Plant journal : for cell and molecular biology, Jan 11, 2018
Respiration in leaves and the continued elevation in the atmospheric CO concentration cause CO -m... more Respiration in leaves and the continued elevation in the atmospheric CO concentration cause CO -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO signal transduction in guard cells. Recently, mutations in the Mitogen-Activated Protein (MAP) kinase, MPK12, have been shown to partially impair CO -induced stomatal closure. Here we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double mutants), completely lack CO -induced stomatal responses and have severely impaired activation of guard cell S-type anion channels in response to elevated CO /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activati...
The atmospheric pollutant ozone (O3 ) is a strong oxidant that causes extracellular reactive oxyg... more The atmospheric pollutant ozone (O3 ) is a strong oxidant that causes extracellular reactive oxygen species (ROS) formation, has significant ecological relevance, and is used here as a non-invasive ROS inducer to study plant signalling. Previous genetic screens identified several mutants exhibiting enhanced O3- sensitivity, but few with enhanced tolerance. We found that loss-of-function mutants in Arabidopsis MLO2, a gene implicated in susceptibility to powdery mildew disease, exhibit enhanced dose-dependent tolerance to O3 and extracellular ROS, but a normal response to intracellular ROS. This phenotype is increased in a mlo2 mlo6 mlo12 triple mutant, reminiscent of the genetic redundancy of MLO genes in powdery mildew resistance. Stomatal assays revealed that enhanced O3 tolerance in mlo2 mutants is not caused by altered stomatal conductance. We explored modulation of the mlo2-associated O3 tolerance, powdery mildew resistance, and early senescence phenotypes by genetic epistasis ...
Stomata can sense the intercellular CO2 concentration (Ci) and water availability and adjust thei... more Stomata can sense the intercellular CO2 concentration (Ci) and water availability and adjust their aperture in order to maintain optimal conditions to photosynthesis under changing environmental conditions. Stomatal movements are regulated by complex network of signaling cascades where reactive oxygen species (ROS) play a key role as signaling component. Recent Advances: Recent research has uncovered several signaling components involved in CO2 and ABA-triggered stomatal guard cell signaling. In addition, we are beginning to understand the complex interactions between different signaling pathways. Under stress-conditions, such as drought, plant close their stomata and subsequent decrease in Ci leads to ROS production through photorespiration and over-reduction of the chloroplast electron transport chain. This reduces plant growth and thus drought may cause severe yield losses for agriculture especially in arid areas. The focus of future research should be drawn towards understanding...
Increasing drought and diminishing freshwater supplies have stimulated interest in developing sma... more Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.
Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is cont... more Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is controlled by stomata, small pores on plant leaves and stems formed by guard cells. Evolution has shaped the morphology and regulatory mechanisms governing stomatal movements to correspond to the needs of various land plant groups over the past 400 million years. Stomata close in response to the plant hormone abscisic acid (ABA), elevated CO2 concentration, and reduced air humidity. Whether the active regulatory mechanisms that control stomatal closure in response to these stimuli are present already in mosses, the oldest plant group with stomata, or were acquired more recently in angiosperms remains controversial. It has been suggested that the stomata of the basal vascular plants, such as ferns and lycophytes, close solely hydropassively. On the other hand, active stomatal closure in response to ABA and CO2 was found in several moss, lycophyte, and fern species. Here, we show that the stom...
The cuticle plays a critical role in plant survival during extreme drought conditions. There are,... more The cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an α/β-hydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated ω-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration.
Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are... more Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2for photosynthesis, loss of water and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata and stomatal CO2-insensitivity phenotypes of theArabidopsis thalianaaccession Cvi-0 to a single amino acid substitution in MAP kinase 12 (MPK12). In parallel we showed that stomatal CO2-insensitivity phenotypes of a mutantcis(CO2-insensitive) were caused by a deletion ofMPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HT1, a central node in guard cell CO2signaling, and that MPK12 can function as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2signaling controls plant water management.
Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control ... more Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control of water-use efficiency [2] in plants. Although the guard-cell-signaling pathway coupling blue light perception to ion channel activity is relatively well understood [3], we know less about the sources of ATP required to drive K(+) uptake [3-6]. Here, we show that triacylglycerols (TAGs), present in Arabidopsis guard cells as lipid droplets (LDs), are involved in light-induced stomatal opening. Illumination induces reductions in LD abundance, and this involves the PHOT1 and PHOT2 blue light receptors [3]. Light also induces decreases in specific TAG molecular species. We hypothesized that TAG-derived fatty acids are metabolized by peroxisomal β-oxidation to produce ATP required for stomatal opening. In silico analysis revealed that guard cells express all the genes required for β-oxidation, and we showed that light-induced stomatal opening is delayed in three TAG catabolism mutants (sdp1...
Ethylene (ET) and jasmonic acid (JA) have opposite effects on ozone (O(3))-induced spreading cell... more Ethylene (ET) and jasmonic acid (JA) have opposite effects on ozone (O(3))-induced spreading cell death; ET stimulates, and is required for the spreading cell death, whereas JA protects tissues. We studied the underlying molecular mechanisms with the O(3)-sensitive, JA-insensitive jasmonate resistant 1 (jar1), and the O(3)-tolerant, ET-insensitive ethylene insensitive 2 (ein2) mutants. Blocking ET perception pharmacologically with norbornadiene (NBD) in jar1, or ET signaling genetically in the jar1 ein2 double mutant prevented the spread of cell death. This suggests that EIN2 function is epistatic to JAR1, and that the JAR1-dependent JA pathway halts oxidative cell death by directly inhibiting ET signaling. JAR1-dependent suppression of the ET pathway was apparent also as increased EIN2-dependent gene expression and ET hypersensitivity of jar1. Physiological experiments suggested that the target of JA is upstream of Constitutive Triple Response 1 (CTR1), but downstream of ET biosynthesis. Gene expression analysis of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated and O(3)-exposed ein2 and jar1 revealed reciprocal antagonism: the EIN2-mediated suppression of the JA pathway. The results imply that the O(3)-induced spreading cell death is stimulated by early, rapid accumulation of ET, which can suppress the protecting function of JA thereby allowing cell death to proceed. Extended spreading cell death induces late accumulation of JA, which inhibits the propagation of cell death through inhibition of the ET pathway.
Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membra... more Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and Q...
Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked b... more Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone (O3 ). Such O3 -pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O3 pulses (200 to 1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic Ca2+ level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.
Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasio... more Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to which 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 on JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2, and npr1 alleles. While stomata in the studied mutants clearly responded to ABA, CO2 , light, and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated comparing with the wild type. Stomatal re-opening 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 the treatments, 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 h and reached a maximum in 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 whole-plant stomatal response to environmental cues in Arabidopsis and tomato.
Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress... more Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 s...
Stomata, small pores on the surfaces of leaves formed by a pair of guard cells, adapt rapidly to ... more Stomata, small pores on the surfaces of leaves formed by a pair of guard cells, adapt rapidly to changes in the environment by adjusting the aperture width. As a long term response, the number of stomata is regulated during stomatal development. The hormone abscisic acid (ABA) regulates both processes. In ABA mediated guard cell signaling the protein kinase OPEN STOMATA1 (OST1) has a central role, as stomatal closure in the ost1 mutant is impaired in response to ABA and to different environmental stimuli. We aimed to dissect the contribution of different ABA-related regulatory mechanisms in determining stomatal conductance, a combination of stomatal density and aperture width, and crossed the ost1 mutant with mutants that either decreased (aba3) or increased (cyp707a1/a3) the concentration of ABA in plants. The double mutant ost1 aba3 had higher stomatal conductance than either parent due to a combination of increased stomatal aperture width and higher stomatal density. In the tripl...
Abstract Optimization between photosynthetic carbon assimilation and stomatal water loss is the k... more Abstract Optimization between photosynthetic carbon assimilation and stomatal water loss is the key in order to breed crops for future climate. We analyzed stomatal conductance (gs), CO2 assimilation rate (Anet), fungal diseases, grain yield and yield components of seven European malting barley genotypes treated with fungicides alone or together with biostimulants in the field over three consecutive seasons. Stomatal conductance, net assimilation rate and grain yield were affected by genotype, treatment and year. We then examined which of these traits are most strongly correlated with yield. Grain yields ranged between 6000-8300 kg ha−1 in 2015 and 2017, but only 3500–6000 kg ha−1 in 2016 due to hotter and drier weather. The benefits of treatments with fungicides or fungicides together with biostimulants were greater in 2016, when the treatments increased yields by 20–21% on average, compared to 0–11% in 2015 and 2017. In 2016, gs and Anet were correlated with grain yield, indicating that in hotter and drier than average season maintaining higher transpiration and photosynthesis resulted in higher yield. In 2015 and 2017, average values of instantaneous gs and Anet were not significantly correlated with grain yield. Pooling all years, longer pre-heading period, higher grain numbers per ha and per ear, larger 1000-kernel weight and higher water use efficiency were associated with higher grain yield, whereas a negative correlation was detected between grain yield and the number of ears per area. Thus, developing fewer productive tillers per area, but more and heavier grains per ear led to higher grain yield. Major barley fungal pathogens had a negative effect on grain yield via shortening of grain filling period.
Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptak... more Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion current...
Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. C... more Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. CO2 elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO2/ABA interaction remain unclear. Two models have been considered: (i) CO2 elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (ii) CO2 signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutants nced3/nced5 and aba2-1 remain responsive to CO2 elevation. Rapid CO2-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays ...
The Plant journal : for cell and molecular biology, Jan 11, 2018
Respiration in leaves and the continued elevation in the atmospheric CO concentration cause CO -m... more Respiration in leaves and the continued elevation in the atmospheric CO concentration cause CO -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO signal transduction in guard cells. Recently, mutations in the Mitogen-Activated Protein (MAP) kinase, MPK12, have been shown to partially impair CO -induced stomatal closure. Here we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double mutants), completely lack CO -induced stomatal responses and have severely impaired activation of guard cell S-type anion channels in response to elevated CO /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activati...
The atmospheric pollutant ozone (O3 ) is a strong oxidant that causes extracellular reactive oxyg... more The atmospheric pollutant ozone (O3 ) is a strong oxidant that causes extracellular reactive oxygen species (ROS) formation, has significant ecological relevance, and is used here as a non-invasive ROS inducer to study plant signalling. Previous genetic screens identified several mutants exhibiting enhanced O3- sensitivity, but few with enhanced tolerance. We found that loss-of-function mutants in Arabidopsis MLO2, a gene implicated in susceptibility to powdery mildew disease, exhibit enhanced dose-dependent tolerance to O3 and extracellular ROS, but a normal response to intracellular ROS. This phenotype is increased in a mlo2 mlo6 mlo12 triple mutant, reminiscent of the genetic redundancy of MLO genes in powdery mildew resistance. Stomatal assays revealed that enhanced O3 tolerance in mlo2 mutants is not caused by altered stomatal conductance. We explored modulation of the mlo2-associated O3 tolerance, powdery mildew resistance, and early senescence phenotypes by genetic epistasis ...
Stomata can sense the intercellular CO2 concentration (Ci) and water availability and adjust thei... more Stomata can sense the intercellular CO2 concentration (Ci) and water availability and adjust their aperture in order to maintain optimal conditions to photosynthesis under changing environmental conditions. Stomatal movements are regulated by complex network of signaling cascades where reactive oxygen species (ROS) play a key role as signaling component. Recent Advances: Recent research has uncovered several signaling components involved in CO2 and ABA-triggered stomatal guard cell signaling. In addition, we are beginning to understand the complex interactions between different signaling pathways. Under stress-conditions, such as drought, plant close their stomata and subsequent decrease in Ci leads to ROS production through photorespiration and over-reduction of the chloroplast electron transport chain. This reduces plant growth and thus drought may cause severe yield losses for agriculture especially in arid areas. The focus of future research should be drawn towards understanding...
Increasing drought and diminishing freshwater supplies have stimulated interest in developing sma... more Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.
Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is cont... more Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is controlled by stomata, small pores on plant leaves and stems formed by guard cells. Evolution has shaped the morphology and regulatory mechanisms governing stomatal movements to correspond to the needs of various land plant groups over the past 400 million years. Stomata close in response to the plant hormone abscisic acid (ABA), elevated CO2 concentration, and reduced air humidity. Whether the active regulatory mechanisms that control stomatal closure in response to these stimuli are present already in mosses, the oldest plant group with stomata, or were acquired more recently in angiosperms remains controversial. It has been suggested that the stomata of the basal vascular plants, such as ferns and lycophytes, close solely hydropassively. On the other hand, active stomatal closure in response to ABA and CO2 was found in several moss, lycophyte, and fern species. Here, we show that the stom...
The cuticle plays a critical role in plant survival during extreme drought conditions. There are,... more The cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an α/β-hydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated ω-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration.
Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are... more Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2for photosynthesis, loss of water and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata and stomatal CO2-insensitivity phenotypes of theArabidopsis thalianaaccession Cvi-0 to a single amino acid substitution in MAP kinase 12 (MPK12). In parallel we showed that stomatal CO2-insensitivity phenotypes of a mutantcis(CO2-insensitive) were caused by a deletion ofMPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HT1, a central node in guard cell CO2signaling, and that MPK12 can function as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2signaling controls plant water management.
Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control ... more Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control of water-use efficiency [2] in plants. Although the guard-cell-signaling pathway coupling blue light perception to ion channel activity is relatively well understood [3], we know less about the sources of ATP required to drive K(+) uptake [3-6]. Here, we show that triacylglycerols (TAGs), present in Arabidopsis guard cells as lipid droplets (LDs), are involved in light-induced stomatal opening. Illumination induces reductions in LD abundance, and this involves the PHOT1 and PHOT2 blue light receptors [3]. Light also induces decreases in specific TAG molecular species. We hypothesized that TAG-derived fatty acids are metabolized by peroxisomal β-oxidation to produce ATP required for stomatal opening. In silico analysis revealed that guard cells express all the genes required for β-oxidation, and we showed that light-induced stomatal opening is delayed in three TAG catabolism mutants (sdp1...
Ethylene (ET) and jasmonic acid (JA) have opposite effects on ozone (O(3))-induced spreading cell... more Ethylene (ET) and jasmonic acid (JA) have opposite effects on ozone (O(3))-induced spreading cell death; ET stimulates, and is required for the spreading cell death, whereas JA protects tissues. We studied the underlying molecular mechanisms with the O(3)-sensitive, JA-insensitive jasmonate resistant 1 (jar1), and the O(3)-tolerant, ET-insensitive ethylene insensitive 2 (ein2) mutants. Blocking ET perception pharmacologically with norbornadiene (NBD) in jar1, or ET signaling genetically in the jar1 ein2 double mutant prevented the spread of cell death. This suggests that EIN2 function is epistatic to JAR1, and that the JAR1-dependent JA pathway halts oxidative cell death by directly inhibiting ET signaling. JAR1-dependent suppression of the ET pathway was apparent also as increased EIN2-dependent gene expression and ET hypersensitivity of jar1. Physiological experiments suggested that the target of JA is upstream of Constitutive Triple Response 1 (CTR1), but downstream of ET biosynthesis. Gene expression analysis of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated and O(3)-exposed ein2 and jar1 revealed reciprocal antagonism: the EIN2-mediated suppression of the JA pathway. The results imply that the O(3)-induced spreading cell death is stimulated by early, rapid accumulation of ET, which can suppress the protecting function of JA thereby allowing cell death to proceed. Extended spreading cell death induces late accumulation of JA, which inhibits the propagation of cell death through inhibition of the ET pathway.
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