Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

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.

Stomatal CO2/bicarbonate sensor consists of two interacting protein kinases, Raf-like HT1 and non-kinase-activity requiring MPK12/MPK4.

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.

Gas Exchange Measurements in Systemic Signaling Studies.

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

The Receptor-like Pseudokinase GHR1 Is Required for Stomatal Closure.

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 currents in Xenopus laevis oocytes and present supporting in silico and in vitro evidence suggesting that GHR1 is an inactive pseudokinase. Biochemical analyses suggested that GHR1-mediated activation of SLAC1 occurs via interacting proteins and that CALCIUM-DEPENDENT PROTEIN KINASE3 interacts with GHR1. We propose that GHR1 acts in stomatal closure as a scaffolding component.

A Dominant Mutation in the HT1 Kinase Uncovers Roles of MAP Kinases and GHR1 in CO2-Induced Stomatal Closure.

Activation of the guard cell S-type anion channel SLAC1 is important for stomatal closure in response to diverse stimuli, including elevated CO2 The majority of known SLAC1 activation mechanisms depend on abscisic acid (ABA) signaling. Several lines of evidence point to a parallel ABA-independent mechanism of CO2-induced stomatal regulation; however, molecular details of this pathway remain scarce. Here, we isolated a dominant mutation in the protein kinase HIGH LEAF TEMPERATURE1 (HT1), an essential regulator of stomatal CO2 responses, in an ozone sensitivity screen of Arabidopsis thaliana The mutation caused constitutively open stomata and impaired stomatal CO2 responses. We show that the mitogen-activated protein kinases (MPKs) MPK4 and MPK12 can inhibit HT1 activity in vitro and this inhibition is decreased for the dominant allele of HT1. We also show that HT1 inhibits the activation of the SLAC1 anion channel by the protein kinases OPEN STOMATA1 and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) in Xenopus laevis oocytes. Notably, MPK12 can restore SLAC1 activation in the presence of HT1, but not in the presence of the dominant allele of HT1. Based on these data, we propose a model for sequential roles of MPK12, HT1, and GHR1 in the ABA-independent regulation of SLAC1 during CO2-induced stomatal closure.

Reactive Oxygen Species in the Regulation of Stomatal Movements.

Guard cells form stomatal pores that optimize photosynthetic carbon dioxide uptake with minimal water loss. Stomatal movements are controlled by complex signaling networks that respond to environmental and endogenous signals. Regulation of stomatal aperture requires coordinated activity of reactive oxygen species (ROS)-generating enzymes, signaling proteins, and downstream executors such as ion pumps, transporters, and plasma membrane channels that control guard cell turgor pressure. Accumulation of ROS in the apoplast and chloroplasts is among the earliest hallmarks of stomatal closure. Subsequent increase in cytoplasmic Ca(2+) concentration governs the activity of multiple kinases that regulate the activity of ROS-producing enzymes and ion channels. In parallel, ROS directly regulate the activity of multiple proteins via oxidative posttranslational modifications to fine-tune guard cell signaling. In this review, we summarize recent advances in the role of ROS in stomatal closure and discuss the importance of ROS in regulation of signal amplification and specificity in guard cells.

Apoplastic and chloroplastic redox signaling networks in plant stress responses.

SIGNIFICANCE: Interplay among apoplastic and chloroplastic redox signaling networks is emerging as a key mechanism in plant stress responses. RECENT ADVANCES: Recent research has revealed components involved in apoplastic and chloroplastic redox signaling. Also, the sequence of events from stress perception, activation of apoplastic reactive oxygen species (ROS) burst through NADPH oxidases, cytoplasmic and chloroplastic Ca(2+)-transients, and organellar redox signals to physiological responses is starting to emerge. Moreover, a functional overlap between light acclimation and plant immunity in photosynthetically active tissues has been demonstrated. CRITICAL ISSUES: Any deviations from the basal cellular redox balance may induce acclimation responses that continuously readjust cellular functions. However, diversion of resources to stress responses may lead to attenuation of growth, and exaggeration of defensive reactions may thus be detrimental to the plant. The ultimate outcome of acclimation responses must therefore be tightly controlled by the redox signaling networks between organellar and apoplastic signaling systems. FUTURE DIRECTIONS: Two major questions still remain to be solved: the sensory mechanism for ROS and the components involved in relaying the signals from the apoplast to the chloroplast. A comprehensive view of regulatory networks will facilitate the understanding on how environmental factors affect the production of phytonutrients and biomass in plants. Translation of such information from model plants to crop species will be at the cutting edge of research in the near future. These challenges give a frame for future studies on ROS and redox regulation of stress acclimation in photosynthetic organisms.