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1. Distinct guard cell–specific remodeling of chromatin accessibility during abscisic acid– and CO ₂ -dependent stomatal regulation

   https://doi.org/10.1073/pnas.2310670120  

   Seller, Charles A; Schroeder, Julian I (December 2023, Proceedings of the National Academy of Sciences)

   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. 

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2. Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana

   https://doi.org/10.1111/tpj.16756  

   Jaafar, Leila; Chen, Yintong; Keynia, Sedighe; Turner, Joseph_A; Anderson, Charles_T (April 2024, The Plant Journal)

   SUMMARY Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized inArabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D‐nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild‐type and cellulose‐deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation. 

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3. PECTATE LYASE LIKE12 patterns the guard cell wall to coordinate turgor pressure and wall mechanics for proper stomatal function in Arabidopsis

   https://doi.org/10.1093/plcell/koab161  

   Chen, Yintong; Li, Wenlong; Turner, Joseph A; Anderson, Charles T (June 2021, The Plant Cell)

   null (Ed.)

   Abstract Plant cell deformations are driven by cell pressurization and mechanical constraints imposed by the nanoscale architecture of the cell wall, but how these factors are controlled at the genetic and molecular levels to achieve different types of cell deformation is unclear. Here, we used stomatal guard cells to investigate the influences of wall mechanics and turgor pressure on cell deformation and demonstrate that the expression of the pectin-modifying gene PECTATE LYASE LIKE12 (PLL12) is required for normal stomatal dynamics in Arabidopsis thaliana. Using nanoindentation and finite element modeling to simultaneously measure wall modulus and turgor pressure, we found that both values undergo dynamic changes during induced stomatal opening and closure. PLL12 is required for guard cells to maintain normal wall modulus and turgor pressure during stomatal responses to light and to tune the levels of calcium cross-linked pectin in guard cell walls. Guard cell-specific knockdown of PLL12 caused defects in stomatal responses and reduced leaf growth, which were associated with lower cell proliferation but normal cell expansion. Together, these results force us to revise our view of how wall-modifying genes modulate wall mechanics and cell pressurization to accomplish the dynamic cellular deformations that underlie stomatal function and tissue growth in plants. 

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4. The Protein Kinase Inhibitor, Tyrphostin AG126, and Its Derivatives Suppress Phosphorylation of Plasma Membrane H+-ATPase and Light-Induced Stomatal Opening

   https://doi.org/10.1093/pcp/pcaf050  

   Kuwayama, Shogo; Hayashi, Maki; Takahashi, Koji; Hayashi, Yuki; Fukatsu, Kohei; Murakami, Kei; Aihara, Yusuke; Sato, Ayato; Kinoshita, Toshinori (May 2025, Plant And Cell Physiology)

   Abstract Phosphorylation of the penultimate residue, threonine (pen-Thr), of plasma membrane (PM) H+-ATPase is essential for its activation and blue light (BL)-induced stomatal opening. However, the regulatory mechanism of action of PM H+-ATPase pen-Thr phosphorylation is not completely understood. Here, we performed screening using a protein kinase inhibitor library and found that tyrphostin AG126 inhibited phosphorylation of PM H+-ATPase pen-Thr in guard cells in response to light and fungal toxin fusicoccin (FC), in addition to inhibition of light- and FC-induced stomatal opening. Analysis of the structure–activity relationship using AG126 derivatives revealed the hydroxyl group at the C-5 position of the compound to be essential for its activity. We further characterized one AG126 derivative, AGD-1, which effectively suppressed BL-induced stomatal opening with a half-inhibitory concentration of 2.0 μM. AGD-1 inhibited PM H+-ATPase pen-Thr phosphorylation in guard cells in response to BL and FC. In addition, AGD-1 suppressed FC-induced PM H+-ATPase pen-Thr phosphorylation in mesophyll cell protoplasts, implying that the effect of AGD-1 is not specific to guard cells. Furthermore, to improve the permeability of AGD-1, we synthesized acetylated AGD-1 (AcAGD-1), which was found to suppress BL- and FC-induced stomatal opening. AcAGD-1 suppressed light-induced PM H+-ATPase pen-Thr phosphorylation, but not Thr881 phosphorylation, in leaf discs, which is important for guard cell PM H+-ATPase activation in addition to pen-Thr phosphorylation. This study identified a novel stomatal opening inhibitor capable of specifically inhibiting PM H+-ATPase pen-Thr phosphorylation. 

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5. Identification of DIR1-Dependant Cellular Responses in Guard Cell Systemic Acquired Resistance

   https://doi.org/10.3389/fmolb.2021.746523  

   David, Lisa; Kang, Jianing; Nicklay, Josh; Dufresne, Craig; Chen, Sixue (December 2021, Frontiers in Molecular Biosciences)

   After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific molecular mechanisms remain elusive. The lipid transfer protein defective in induced resistance 1 (DIR1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Here, we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1-1 mutant during SAR. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were also changed in guard cells of dir1-1 compared to the wild type. Identification of guard cell-specific SAR-related molecules may lead to new avenues of genetic modification/molecular breeding for disease-resistant plants. 

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