Search

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.

The continuing rise in the atmospheric carbon dioxide (CO2) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO2 sensor remains unknown. Here, we show that elevated CO2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO2, HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO2. Dominant HT1 mutants abrogate the CO2/bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO2 sensor function and CO2-triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO2/bicarbonate sensor upstream of the CBC1 kinase in plants.

Summary: Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood.We used an ozone‐sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation.High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata‐closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP‐l‐fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP‐l‐Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi‐localized fucosylation events. However, impaired fucosylation of xyloglucan, N‐linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants.Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l‐fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole‐plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant–water relations. [ABSTRACT FROM AUTHOR]

Summary: Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co‐receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear.We took a gain‐of‐function approach to study receptor‐specific functionality. First, we introduced activating mutations that enforce active ABA‐bound receptor conformation. We then transformed Arabidopsis ABA‐deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype.Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism.We propose that ABA signaling – from perception to response – flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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). Significance Statement: Jasmonic acid (JA) and salicylic acid (SA) can promote stomatal closure; however, the roles of these hormones in stomatal responses to environmental cues remain unclear. Here, we show that stomatal responsiveness to CO2, light intensity, abscisic acid, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended on JA and SA. [ABSTRACT FROM AUTHOR]

Summary: Low concentrations of CO2 cause stomatal opening, whereas [CO2] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+‐binding proteins that function in the stomatal CO2 response remain unknown.Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed.We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2, light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2‐induced stomatal closure and, unexpectedly, also in low CO2‐induced stomatal opening. Furthermore, K+‐uptake‐channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targeted cbl1/4/5/8/9 quintuple mutant plants.Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response. [ABSTRACT FROM AUTHOR]

The article discusses the elevated carbon dioxide (CO2) triggers interaction of the Mitogen-activated protein (MAP) kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. It discusses about identifying dominant active HT1 mutants that cause insensitivity to elevated CO2; and the presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO2/bicarbonate sensor upstream of the CBC1 kinase in plants.

There is a need for ground-breaking technologies to boost crop yield, both grains and biomass, and their processing into economically competitive materials. Novel cereals with enhanced photosynthesis and assimilation of greenhouse gasses, such as carbon dioxide and ozone, and tailored straw suitable for industrial manufacturing, open a new perspective for the circular economy. Here we describe the vision, strategies, and objectives of BEST-CROP, a Horizon-Europe and United Kingdom Research and Innovation (UKRI) funded project that relies on an alliance of academic plant scientists teaming up with plant breeding companies and straw processing companies to use the major advances in photosynthetic knowledge to improve barley biomass and to exploit the variability of barley straw quality and composition. We adopt the most promising strategies to improve the photosynthetic properties and ozone assimilation capacity of barley: (i) tuning leaf chlorophyll content and modifying canopy architecture; (ii) increasing the kinetics of photosynthetic responses to changes in irradiance; (iii) introducing photorespiration bypasses; (iv) modulating stomatal opening, thus increasing the rate of carbon dioxide fixation and ozone assimilation. We expect that by improving our targeted traits we will achieve increases in aboveground total biomass production without modification of the harvest index, with added benefits in sustainability via better resource-use efficiency of water and nitrogen. In parallel, the resulting barley straw is tailored to: (i) increase straw protein content to make it suitable for the development of alternative biolubricants and feed sources; (ii) control cellulose/lignin contents and lignin properties to develop straw-based construction panels and polymer composites. Overall, by exploiting natural- and induced-genetic variability as well as gene editing and transgenic engineering, BEST-CROP will lead to multi-purpose next generation barley cultivars supporting sustainable agriculture and capable of straw-based applications., (© 2025 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g. , comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

The amino acid cysteine plays a major role in plant response to abiotic stress by being the donor of elemental sulfur for the sulfuration of the molybdenum cofactor, otherwise the last step of ABA biosynthesis, the oxidation of abscisic aldehyde, is inactivated. Additionally, cysteine serves as a precursor for the biosynthesis of glutathione, the reactive oxygen species scavenger essential for redox status homeostasis during stress. Cysteine is generated by the sulfate reductive pathway where sulfite oxidase (SO; EC 1.8.3.1) is an important enzyme in the homeostasis of sulfite levels (present either as a toxic intermediate in the pathway or as a toxic air pollutant that has penetrated the plant tissue via the stomata). SO is localized to the peroxisomes and detoxifies excess sulfite by catalyzing its oxidation to sulfate. Here we show a kinetic assay that relies on fuchsin colorimetric detection of sulfite, a substrate of SO activity. This SO assay is highly specific, technically simple, and readily performed in any laboratory.5'-adenylylsulfate (APS) reductase (APR, E.C. 1.8.4.9) enzyme regulates a crucial step of sulfate assimilation in plants, algae and some human pathogens. The enzyme is upregulated in response to oxidative stress induced by abiotic stresses, such as salinity and hydrogen peroxide, to generate sulfite an intermediate for cysteine generation essential for the biosynthesis of glutathione, the hydrogen peroxide scavenger. Here we present two robust, sensitive, and simple colorimetric methods of APR activity based on sulfite determination by fuchsin.Sulfite reductase (SiR) is one of the key enzymes in the primary sulfur assimilation pathway. It has been shown that SiR is an important plant enzyme for protection plant against sulfite toxicity and premature senescence. Here we describe two methods for SiR activity determination: a kinetic assay using desalted extract and an in-gel assay using crude extract.Due to the energetically favorable equilibrium, sulfurtransferase (ST) activity measured as sulfite generation or consumption. Sulfite-generating ST activity is determined by colorimetric detection of SCN - formation at 460 nm as the red Fe(SCN) 3 complex from cyanide and thiosulfate using acidic iron reagent. Sulfite-consuming (MST) activity is detected as sulfite disappearance in the presence of thiocyanate (SCN - ) or as SCN - disappearance. To abrogate interfering SO activity, total ST activities is detected by inhibiting SO activity with tungstate.

In response to oxidative stress the biosynthesis of the ROS scavenger, glutathione is induced. This requires the induction of the sulfate reduction pathway for an adequate supply of cysteine, the precursor for glutathione. Cysteine also acts as the sulfur donor for the sulfuration of the molybdenum cofactor, crucial for the last step of ABA biosynthesis. Sulfate and sulfite are, respectively, the precursor and intermediate for cysteine biosynthesis and there is evidence for stress-induced sulfate uptake and further downstream, enhanced sulfite generation by 5'-phosphosulfate (APS) reductase (APR, EC 1.8.99.2) activity. Sulfite reductase (SiR, E.C.1.8.7.1) protects the chloroplast against toxic levels of sulfite by reducing it to sulfide. In case of sulfite accumulation as a result of air pollution or stress-induced premature senescence, such as in extended darkness, sulfite can be oxidized to sulfate by sulfite oxidase. Additionally sulfite can be catalyzed to thiosulfate by sulfurtransferases or to UDP-sulfoquinovose by SQD1, being the first step toward sulfolipid biosynthesis.Determination of total sulfur in plants can be accomplished using many techniques such as ICP-AES, high-frequency induction furnace, high performance ion chromatography, sulfur combustion analysis, and colorimetric titration. Here we describe a total sulfur detection method in plants by elemental analyzer (EA). The used EA method is simple, sensitive, and accurate, and can be applied for the determination of total S content in plants.Sulfate anions in the soil are the main source of sulfur, required for normal growth and development, of plants. Plants take up sulfate ions from the soil, which are then reduced and incorporated into organic matter. Plant sulfate content can be determined by ion chromatography with carbonate eluents.Sulfite is an intermediate in the reductive assimilation of sulfate to the essential amino acids cysteine and methionine, and is cytotoxic above a certain threshold if not rapidly metabolized and can wreak havoc at the cellular and whole plant levels. Plant sulfite content affects carbon and nitrogen homeostasis Therefore, methods capable of determining sulfite levels in plants are of major importance. Here we present two robust laboratory protocols which can be used for sulfite detection in plants.Thiosulfate is an essential sulfur intermediate less toxic than sulfite which is accumulating in plants in response to sulfite accumulation. The complexity of thiosulfate detection is linked to its chemical properties. Here we present a rapid, sensitive, and accurate colorimetric method based on the enzymatic conversion of thiosulfate to thiocyanate.The plant sulfolipid sulfoquinovosyldiacylglycerol (SQDG) accounts for a large fraction of organic sulfur in the biosphere. Aside from sulfur amino acids, SQDG represents a considerable sink for sulfate in plants and is the only sulfur-containing anionic glycerolipid that is found in the photosynthetic membranes of plastids. We present the separation of sulfolipids from other fatty acids in two simple ways: by one- and two-dimensional thin-layer chromatography.