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Abstract Stomatal pores in land plants rapidly and reversibly open and close in response to diurnal changes in leaf carbon dioxide (CO2) concentration. Studies have suggested that CO2 is sensed by guard cells with relevant amplifying contributions from mesophyll tissue. CO2 concentration changes trigger rapid signal transduction events involving protein phosphorylation in guard cells. Moreover, molecular crosstalk and physiological interactions of the stomatal CO2 response with other environmental conditions and stimuli, including light, temperature, drought, and abscisic acid, are reviewed here. Genetic studies have revealed several key genes and provided important insights into the stomatal CO2 sensors and signal transduction mechanisms. The primary CO2/HCO3− sensor in Arabidopsis guard cells was recently identified. Quantitative trait locus (QTL) analyses have shown that early guard cell CO2 signal transduction components regulate water use efficiency (WUE). In this review, we describe the molecular details of stomatal CO2 sensing by CO2/HCO3−-induced interaction of two protein kinases, the HIGH LEAF TEMPERATURE 1 Raf-like kinase and the MPK4/MPK12 mitogen-activated protein kinases. The evolutionary emergence of, physiological relevance of, and potential for improvement of WUE of plants via the stomatal CO2 response and open questions in this research field are discussed. 

Abstract In plants, both developmental processes and environmental responses are spatiotemporally regulated by an assembly of signaling molecules such as hormones, secondary metabolites, and ions. The ability of these signaling molecules to move within and across plant tissues is essential for various developmental cues. However, the characterization of transported signaling molecules and their translocation mechanisms is difficult due to the functional redundancy of plant genomes and shortcomings in methodologies. Here, we report our development of the Multi Targeted AmiRNA Cell type–specific Transportome-scale (mTACT) toolbox, which can be used to reveal phenotypic plasticity in plants. mTACT is based on a large set of artificial microRNAs (amiRNAs), each designed to optimally target multiple members of a particular gene family encoding transporter proteins. In total, the mTACT toolbox includes 5,565 amiRNAs, targeting 81.7% of the Arabidopsis (Arabidopsis thaliana) transportome. The amiRNA library can be driven under 12 cell type–specific promoters, allowing the design of spatial-specific genetic screens. mTACT is further divided into 8 sublibraries of amiRNAs targeting a functionally defined protein class. A proof-of-concept screen validated the mTACT approach by identifying phenotypes linked to both known and unidentified genes. With the ability to overcome functional redundancy in a transportome-scale, cell type–specific manner, the mTACT toolbox will allow the plant research community to study previously hidden genetic factors required for long- and short-distance translocation of signaling molecules. 

Summary Plants integrate environmental stimuli to optimize photosynthesis vs water loss by controlling stomatal apertures. However, stomatal responses to temperature elevation and the underlying molecular genetic mechanisms remain less studied.We developed an approach for clamping leaf‐to‐air vapor pressure difference (VPD_leaf) to fixed values, and recorded robust reversible warming‐induced stomatal opening in intact plants. We analyzed stomatal temperature responses of mutants impaired in guard cell signaling pathways for blue light, abscisic acid (ABA), CO₂, and the temperature‐sensitive proteins, Phytochrome B (phyB) and EARLY‐FLOWERING‐3 (ELF3).We confirmed thatphot1‐5/phot2‐1leaves lacking blue‐light photoreceptors showed partially reduced warming‐induced stomatal opening. Furthermore, ABA‐biosynthesis, phyB, and ELF3 were not essential for the stomatal warming response. Strikingly,Arabidopsis(dicot) andBrachypodium distachyon(monocot) mutants lacking guard cell CO₂sensors and signaling mechanisms, includinght1,mpk12/mpk4‐gc, andcbc1/cbc2abolished the stomatal warming response, suggesting a conserved mechanism across diverse plant lineages. Moreover, warming rapidly stimulated photosynthesis, resulting in a reduction in intercellular (CO₂). Interestingly, further enhancing heat stress caused stomatal opening uncoupled from photosynthesis.We provide genetic and physiological evidence that the stomatal warming response is triggered by increased CO₂assimilation and stomatal CO₂sensing. Additionally, increasing heat stress functions via a distinct photosynthesis‐uncoupled stomatal opening pathway. 

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei fromArabidopsis thalianaplants to examine whether the physiological signals, ABA and CO₂(carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type–specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell–specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO₂induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO₂had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance. 

Guard cells, which regulate stomatal apertures in plants, possess a sophisticated mechanism for regulating turgor pressure. The outward-rectifying “K⁺_out” channel GORK, expressed in guard cells of the plantArabidopsis thaliana, is a central component that promotes stomatal closure by releasing K⁺to the extracellular space, thereby lowering turgor pressure. To date, the structural basis underlying the regulation of the K⁺transport activity of GORK is unclear. Using cryo-EM, we determined the structures of the GORK outward-rectifying K⁺channel with a resolution of 3.16 to 3.27 Å in five distinct conformations that differ significantly in their C-terminal cyclic nucleotide binding domain (CNBD) and ankyrin repeat (ANK) domain. The C-linker connects the transmembrane domains to the C-terminal domains, i.e., CNBD, CNBD–Ankyrin bridge, and ANK. The structural changes and interactions in the C-linker determine whether the closed state of GORK is closer to the preopen state or in a more removed state from the open state of the channel. In particular, interconversion in the short sequence within the CNBD–Ankyrin bridge plays a decisive role in this determination. This region forms an α-helix in the preopened state, while it adopts a nonhelical structure in further distant closed states. The dynamics of the cytosolic region strongly suggest that the K⁺channel activity of GORK is regulated by cytosolic signaling factors during stomatal closure. 

Abstract Protein kinase dynamics play key roles in regulation of cell differentiation, growth, development and in diverse cell signaling networks. Protein kinase sensors enable visualization of protein kinase activity in living cells and tissues in time and space. These sensors have therefore become important and powerful molecular tools for investigation of diverse kinase activities and can resolve long-standing and challenging biological questions. In the present Update, we review new advanced approaches for genetically encoded protein kinase biosensor designs developed in animal systems together with the basis of each biosensor’s working principle and components. In addition, we review recent first examples of real time plant protein kinase activity biosensor development and application. We discuss how these sensors have helped to resolve how stomatal signal transduction in response to elevated CO2 merges with abscisic acid signaling downstream of a resolved basal SnRK2 kinase activity in guard cells. Furthermore, recent advances, combined with the new strategies described in this Update, can help deepen the understanding of how signaling networks regulate unique functions and responses in distinct plant cell types and tissues and how different stimuli and signaling pathways can interact.