Artificial cellulosic leaf with adjustable enzymatic CO2 sequestration capability.

Developing artificial leaves to address the environmental burden of CO2 is pivotal for advancing our Net Zero Future. In this study, we introduce EcoLeaf, an artificial leaf that closely mimics the characteristics of natural leaves. It harnesses visible light as its sole energy source and orchestrates the controlled expansion and contraction of stomata and the exchange of petiole materials to govern the rate of CO2 sequestration from the atmosphere. Furthermore, EcoLeaf has a cellulose composition and mechanical strength similar to those of natural leaves, allowing it to seamlessly integrate into the ecosystem during use and participate in natural degradation and nutrient cycling processes at the end of its life. We propose that the carbon sequestration pathway within EcoLeaf is adaptable and can serve as a versatile biomimetic platform for diverse biogenic carbon sequestration pathways in the future.

A network-based modeling framework reveals the core signal transduction network underlying high carbon dioxide-induced stomatal closure in guard cells.

Stomata are pores on plant aerial surfaces, each bordered by a pair of guard cells. They control gas exchange vital for plant survival. Understanding how guard cells respond to environmental signals such as atmospheric carbon dioxide (CO2) levels is not only insightful to fundamental biology but also relevant to real-world issues of crop productivity under global climate change. In the past decade, multiple important signaling elements for stomatal closure induced by elevated CO2 have been identified. Yet, there is no comprehensive understanding of high CO2-induced stomatal closure. In this work, we assemble a cellular signaling network underlying high CO2-induced stomatal closure by integrating evidence from a comprehensive literature analysis. We further construct a Boolean dynamic model of the network, which allows in silico simulation of the stomatal closure response to high CO2 in wild-type Arabidopsis thaliana plants and in cases of pharmacological or genetic manipulation of network nodes. Our model has a 91% accuracy in capturing known experimental observations. We perform network-based logical analysis and reveal a feedback core of the network, which dictates cellular decisions in closure response to high CO2. Based on these analyses, we predict and experimentally confirm that applying nitric oxide (NO) induces stomatal closure in ambient CO2 and causes hypersensitivity to elevated CO2. Moreover, we predict a negative regulatory relationship between NO and the protein phosphatase ABI2 and find experimentally that NO inhibits ABI2 phosphatase activity. The experimental validation of these model predictions demonstrates the effectiveness of network-based modeling and highlights the decision-making role of the feedback core of the network in signal transduction. We further explore the model's potential in predicting targets of signaling elements not yet connected to the CO2 network. Our combination of network science, in silico model simulation, and experimental assays demonstrates an effective interdisciplinary approach to understanding system-level biology.

PIF transcriptional regulators are required for rhythmic stomatal movements.

Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K+ channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K+ intake driving stomata opening.

Heat Waves Coupled with Nanoparticles Induce Yield and Nutritional Losses in Rice by Regulating Stomatal Closure.

The frequency, duration, and intensity of heat waves (HWs) within terrestrial ecosystems are increasing, posing potential risks to agricultural production. Cerium dioxide nanoparticles (CeO2 NPs) are garnering increasing attention in the field of agriculture because of their potential to enhance photosynthesis and improve stress tolerance. In the present study, CeO2 NPs decreased the grain yield, grain protein content, and amino acid content by 16.2, 23.9, and 10.4%, respectively, under HW conditions. Individually, neither the CeO2 NPs nor HWs alone negatively affected rice production or triggered stomatal closure. However, under HW conditions, CeO2 NPs decreased the stomatal conductance and net photosynthetic rate by 67.6 and 33.5%, respectively. Moreover, stomatal closure in the presence of HWs and CeO2 NPs triggered reactive oxygen species (ROS) accumulation (increased by 32.3-57.1%), resulting in chloroplast distortion and reduced photosystem II activity (decreased by 9.4-36.4%). Metabolic, transcriptomic, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that, under HW conditions, CeO2 NPs activated a stomatal closure pathway mediated by abscisic acid (ABA) and ROS by regulating gene expression (PP2C, NCED4, HPCA1, and RBOHD were upregulated, while CYP707A and ALMT9 were downregulated) and metabolite levels (the content of γ-aminobutyric acid (GABA) increased while that of gallic acid decreased). These findings elucidate the mechanism underlying the yield and nutritional losses induced by stomatal closure in the presence of CeO2 NPs and HWs and thus highlight the potential threat posed by CeO2 NPs to rice production during HWs.

Simultaneous and Independent Abaxial and Adaxial Gas Exchange Measurements.

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

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

OPEN STOMATA 1 phosphorylates CYCLIC NUCLEOTIDE-GATED CHANNELs to trigger Ca2+ signaling for abscisic acid-induced stomatal closure in Arabidopsis.

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana.

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H2O2, as indicated by fluorescence probes, BES-So-AM and CM-H2DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H2O2 scavenger) confirmed that both the superoxide and H2O2 were requisites for stomatal closure. Co-localization of the superoxide and H2O2 in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H2O2 generation in mitochondria. In contrast, MD elevated superoxide/H2O2 levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.

Stomatal development: NRPM proteins in dynamic localization of ERECTA receptor.

Dynamic cellular localization of receptors is key to the perception of their peptide ligands and the activation of downstream signaling pathways. A new study identifies NRPMs as novel regulators of ERECTA receptor localization and stomatal formation downstream of the EPF1/EPF2 peptide ligands and upstream of the YDA MAPK cascade.

Phosphorylation of plasma membrane H+-ATPase Thr881 participates in light-induced stomatal opening.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1-9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana.

Light-induced stomatal opening requires phosphorylation of the C-terminal autoinhibitory domain of plasma membrane H+-ATPase.

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.

A charged existence: A century of transmembrane ion transport in plants.

If the past century marked the birth of membrane transport as a focus for research in plants, the past 50 years has seen the field mature from arcane interest to a central pillar of plant physiology. Ion transport across plant membranes accounts for roughly 30% of the metabolic energy consumed by a plant cell, and it underpins virtually every aspect of plant biology, from mineral nutrition, cell expansion, and development to auxin polarity, fertilization, plant pathogen defense, and senescence. The means to quantify ion flux through individual transporters, even single channel proteins, became widely available as voltage clamp methods expanded from giant algal cells to the fungus Neurospora crassa in the 1970s and the cells of angiosperms in the 1980s. Here, I touch briefly on some key aspects of the development of modern electrophysiology with a focus on the guard cells of stomata, now without dispute the premier plant cell model for ion transport and its regulation. Guard cells have proven to be a crucible for many technical and conceptual developments that have since emerged into the mainstream of plant science. Their study continues to provide fundamental insights and carries much importance for the global challenges that face us today.

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

SCAB1 coordinates sequential Ca2+ and ABA signals during osmotic stress induced stomatal closure in Arabidopsis.

Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability. Stomata are gateways of transpiration and gas exchange, the swift adjustment of stomatal aperture has a strong influence on plant drought resistance. Despite intensive investigations of stomatal closure during drought stress in past decades, little is known about how sequential signals are integrated during complete processes. Here, we discovered that the rapid Ca2+ signaling and subsequent abscisic acid (ABA) signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana, while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1) is the molecular switch for this entire process. During the early stage of osmotic shock responses, swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3 (CPK3) and CPK6. The phosphorylation restrained the microfilament binding affinity of SCAB1, which bring about the F-actin disassembly and stomatal closure initiation. As the osmotic stress signal continued, both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly. We further found that ABA signaling is indispensable for these attenuations, which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure. Notably, the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure. Taken together, our results support a model in which SCAB1 works as a molecular switch, and directs the microfilament rearrangement through integrating the sequentially generated Ca2+ and ABA signals during osmotic stress induced stomatal closure.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells.

Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca2+) levels, the interdependence between protons (H+) and Ca2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin HcKCR2 from the pseudofungus Hyphochytrium catenoides, which operates as a H+ conductive, Ca2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of HcKCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca2+ release from the endoplasmic reticulum. A H+-induced cytosolic Ca2+ signal results in membrane depolarization through the activation of Ca2+-dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H+-induced Ca2+ release mechanism in plant cells and establishes HcKCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca2+ signaling.

Singlet oxygen induces cell wall thickening and stomatal density reducing by transcriptome reprogramming.

Singlet oxygen (1O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants.