Stomata

Stomata provide a framework to study the fundamental processes of plants at different organizational levels, from molecules and cells to whole plants and ecosystems. And across vast temporal scales, from milliseconds to millennia. Our lab focuses on the molecules and the mechanisms that make and pattern stomata, but this work is enriched by collaborations: with ecophysiologists, who used tools derived from the molecular-scale studies to improve organismal-scale models for photosynthetic activity; with evolutionary biologists, who, taking advantage of stomatal preservation and sequencing of historic DNA, have been able to track how stomatal genes and developmental patterns change, and how this might link to climate change in the Anthropocene. In this talk, I will highlight open questions and potential places of synergy for our community to explore in the pursuit of an integrated understanding of stomatal biology.

When did stomata first evolve? What was their function in the first land plants? Stomata are a fundamental aspect of the land plant body plan, yet understanding their evolution requires a careful appraisal of the fossil record within a robust phylogenetic framework. I will discuss how we have combined phylogenomics, comparative genomics, and palaeontology with analyses of physiology and gene expression to reveal the origins and trajectory of stomatal evolution.

Stomata will play a critical role in determining how the world’s terrestrial vegetation responds to our changing climate. In this talk I will review some recent advances in our ability to predict stomatal behaviour under rising CO2 concentrations, rising temperature, heatwaves, and drought. Many new insights have been gained through the use of optimisation approaches to predict stomatal conductance, and I will briefly survey some of these models. I will also discuss “non-optimal” stomatal behaviour: what can we learn when stomata don’t behave as optimisation models predict?

Most plants have all or most of their stomata in the lower (abaxial) leaf side, whereas grasses and many other herbs, including the model plant Arabidopsis thaliana (Arabidopsis), have high stomatal numbers also on the upper (adaxial) leaf surface. Most studies on stomatal development and guard cell signalling have focused on abaxial stomata and very little is known on the formation and role of adaxial stomata in plants. Stomatal ratio describes the distribution of stomata between upper and lower leaf surfaces. How stomatal ratio is determined in plants remains unclear. Our recent work indicates that: i) mechanisms that govern stomatal development in the adaxial and abaxial epidermis in Arabidopsis are at least partly different, ii) adaxial and abaxial stomatal development and conductance respond differently to changes in environmental conditions, leading to changes in stomatal ratio and stomatal conductance ratio; and iii) stomatal ratio is positively related with yield in tomatoes. Thus, understanding the mechanisms of adaxial stomatal development and how stomatal ratio is adjusted in plants can help to breed crops with improved yield.

Stomatal guard cells are encased in strong, flexible cell walls that underpin their dynamic responses to signaling cues, but how these walls are assembled with these properties is incompletely understood. Here, we explored how the properties of guard cell walls change during stomatal maturation in Arabidopsis thaliana and result in stomatal complexes that open and close efficiently. We established milestones for stomatal maturation, when the stomatal pore and guard cells enlarge with distinct kinetics, and found that although guard cell walls thicken during maturation, they become more mechanically anisotropic and achieve stomatal opening with smaller changes in turgor pressure and less energy input. We also found that cellulose is required for normal stomatal maturation and that both cellulose and pectins are critical for mechanical anisotropy and efficient stomatal opening in mature guard cells. Based on their molecular architectures, we developed a multi-scale model that recapitulates the biomechanics of wild type and mutant guard cell walls. Finally, we used cell ablation and automated cell segmentation in both Arabidopsis and grass stomatal complexes to find that cells adjacent to guard cells constrain their motions in unexpected ways. Together, these data functionally connect the molecular composition and structure of guard cell walls to their biomechanical properties and the efficiency of stomatal dynamics.

How does the environment affect cell fate? My research group employs stomatal development in plants as a model system to explore the influence of external signals on cell fate decisions at the molecular and cell lineage level. The development of stomata—the surface pores on plants critical for gas and water vapour exchange—represents an excellent model because stomatal production is highly responsive to diverse environmental stimuli and its regulation has strong implications to agricultural production. In my talk, I will discuss our recent efforts in addressing how external signals, such as drought, modulate stomatal formation. By focusing on the stem cell-like stomatal precursor cells, we elucidated the mechanism underlying these responses and identified key nodes in the plasticity of stomatal production. Our works highlight how the production of a specific cell type can be controlled and coordinated, and how the insights may be applied to improve plant fitness.

Our work is focused on understanding the signalling mechanisms that mediate plant developmental changes in response to environmental signals. Stomata, the microscopic pores on the leaf surface, are an excellent model for examining how environmental signals modulate plant development. Factors such as light quantity and quality as well as atmospheric carbon dioxide have a major impact on stomatal development. Using a combination of genetic and molecular tools our work has demonstrated that plant photoreceptors, significantly phyB, play a critical role in regulating stomatal development in response to environmental signals. However, photoreceptor signalling is insufficient to explain some light responses and here, photosynthetic regulation plays a major role. This pathway targets key stomatal regulators and in combination with photoreceptor control, allows for fine tuning of stomatal development under different conditions.

As a strategy for water use efficiency and biomass production, accelerating stomata has proven viable through synthetic optogenetics and mutations that enhance guard cell K+ flux. Here I outline some of our discoveries from cryoEM analysis of the Arabidopsis thaliana GORK K+ channel structure. I examine features of GORK gating mutants and the natural variant GROK from the related model C4 species Gynandropsis gynandra. These molecular features contribute to gating, facilitate K+ flux, and speed stomatal movements. Some will clearly translate to crop species to enhance water use efficiency and biomass gains. For GROK, the evidence also speaks to the puzzle of how C4 plant have evolved mechanisms that enhance water use efficiency and growth under stress.

The role of sucrose for stomatal movement regulation has long been debated, with several controversial studies and theories. Here we demonstrated that sucrose concentration at the leaf apoplast underpin the diel course of tobacco stomatal conductance (gs), in which the daily stomatal opening and closure were associated with low and high concentration of apoplastic sucrose, respectively. In agreement with this, exogenously applied sucrose increased the speediness of both stomatal opening and closure in a concentration-dependent manner. We further showed that the light-induced stomatal opening is closely associated to the dynamic of sucrose and organic acids within guard cells. Interestingly, these sucrose-mediated stomatal responses were drastically reduced in plants with diminished capacity to import sucrose to their guard cells, highlighting that sucrose importation to these cells is important to modulate the magnitude of both stomatal opening and closure. Modelling analysis highlights that the metabolism of the apoplast rather than the leaf is the major determinant of the daily gs. Our results collectively indicate that sucrose is a master regulator of the daily gs, being capable of inducing and accelerating both stomatal opening and closure in a concentration and location of accumulation dependent manner.

Stomatal movements are associated with pH changes in guard cells. Several authors have demonstrated that cytosolic alkalinization preceded the rise in ROS or Ca2+of guard cells. In contrast, a few reports suggest that the increase in cytosolic pH follows the elevated ROS or Ca2+, suggesting that cytosolic pH rise may not always be an early event. The components, such as ROS, Ca2+, and Ca2+-dependent protein kinases, converge to modulate ion channels, promote ion efflux from guard cells and promote stomatal closure. We propose a hypothetical model to integrate the event of pH rise with other signalling components and explain the argument that cytosolic alkalization can occur downstream or upstream of ROS or Ca2+-rise. Changes in guard cell pH can occur when ATPases are modulated. Stomatal closure and guard cell pH rise are compromised in mutants deficient in vacuolar H+-ATPase (V-ATPase), pointing out the role of V-ATPase. My talk attempts to consider arguments for and against the significance of cytosolic alkalinization in guard cells. Stomatal guard cells are promising model systems for further research into this intriguing topic of cytosolic pH change during stomatal closure.

Guard cell starch metabolism plays a central role in the regulation of stomatal movements in response to light, elevated ambient CO2 and potentially other abiotic and biotic factors. In my talk, I will discuss how various guard cell signal transduction pathways converge to promote rearrangements in guard cell starch metabolism for efficient stomatal responses - an essential physiological process that sustains plant productivity and stress tolerance. I will argue that manipulation of guard cell starch dynamics may improve stomatal behaviour under changing environmental conditions.

Each formed by a pair of guard cells, stomata are microscopical pores that play fundamental roles in plant photosynthesis, water use efficiency and stress adaptation. The simple stomatal structure and specialised guard cell anatomy enable plants to respond and adapt to environmental changes rapidly. To identify genetic determinants involved in stomatal formation and maturation, we took advantage of forward genetic approaches and performed focused mutant screens. Here, we report the isolation and characterization of two mutants, lds1 and dsm1. LIPID DROPLETS AND STOMATA 1 (LDS1)/RABC1 (At1g43890) encodes a member of the Rab GTPase family that is involved in regulating lipid droplets (LD) dynamics. The expression of LDS1/RABC1 is coordinated with the different phases of stomatal development. RABC1 physically interacts with SEIPIN2/3, two orthologues of mammalian Seipin, which function in the formation of LD. Disruption of RABC1, RABC1GEF1, or SEIPIN2/3 resulted in aberrantly large LD, severe defects in guard cell vacuole morphology, stomatal movements. DEFORMED STOMATA 1 (DSM1) 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. Furthermore, we demonstrated that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification. Our work provides necessary insight into the regulatory mechanisms of stomatal morphogenesis and function in Arabidopsis.

Recent advances showed that carbohydrate metabolism is essential for light induced stomatal opening. Starch in guard cells degrades quickly upon light exposure. Phosphoproteomic analysis revealed that ß-amylase 1 (BAM1), which is specifically expressed in guard cells and responsible for starch degradation, is potential target of Target of Rapamycin (TOR). TOR kinase stabilizes BAM1 by directly phosphorylating Serine 31, thereby promoting guard cell starch degradation and stomatal opening. Meanwhile, stomata is high energy demanded organ and possesses higher ability of respiration, leading to guard cell specific accumulation of hydrogen peroxide (H2O2) under unstressed condition. Guard cell specific accumulated H2O2 is required for light induced starch degradation and stomatal opening. This phenomenon is conserved among plant species with different stomata types and basal vascular plants. H2O2 promotes guard cell starch degradation and stomatal opening through KIN10, the catalytic subunit of SnRK1. KIN10 prefers nuclear localization caused by H2O2 accumulation in guard cells and phosphorylates bZIP30 transcription factor promoting the expression of BAM1. bZIP30 forms transcriptional complex with BZR1, the core transcription factor of Brassinosteroid signaling, through the KIN10 dependent phosphorylation of bZIP30 and BZR1 oxidation. And the molecular mechanism of KIN10-bZIP30-BZR1 module also exists in Selaginella doederleinii. Our study unveils that metabolic statues regulates light induce stomatal opening through TOR and SnRK1 signaling.

In my talk, I would like to honour the work of Jaakko Kangasjärvi, who passed away on May 14, 2024. Jaakko was an excellent colleague, an inspiring mentor, and he made a major contribution to several fields of plant biology, including stomatal regulation. Back in early 90’s, after returning from US, Jaakko initiated an ozone-sensitivity-based mutant screen to study hormonal regulation of ROS-induced cell death. This screen also identified guard cell anion channel SLAC1; a target of cellular signalling systems triggered by majority of stimuli affecting stomatal closure. It also helped to define interaction between MPK12/4:HT1 kinases as a molecular switch regulating CO2-induced stomatal movements, and clarified the function of other important guard cell proteins, such as GHR1, OST1 etc. Roughly 10 years ago when we initiated Helsinki-Tartu joint mutant screen, Jaakko’s main aim was to identify proteins involved in ozone sensing in guard cells. The screen was again based on ozone sensitivity but designed as saturating and focused on finding proteins involved in stomatal regulation by environmental factors. Now when we are ready to report early steps of ozone sensing in guard cells, he is unfortunately not among us anymore.

Cold stress is a major abiotic stress that threatens maize (Zea mays L.) production worldwide. Using reverse genetics approach, we identified type-A Response Regulator 1 (ZmRR1) as a positive regulator, and transcription factor ZmbZIP68 and tonoplast intrinsic protein TIP4;3 as negative regulators of maize tolerance, respectively. Th protein stability of ZmRR1 and ZmbZIP68 is controlled by ZmMPK8-mediated phosphorylation. The natural variations of ZmRR1 occur at its phosphorylation site, resulting in the divergence of protein stability and chilling tolerance. The ZmbZIP68 locus was a target of selection during early domestication. An Indel polymorphism in the ZmbZIP68 promoter resulted in the differential expression of ZmbZIP68 between maize and its wild ancestor teosinte. TIPs are a subfamily of aquaporins in plants. Here, we report that TIP family proteins are involved in maize cold tolerance. The expression of most TIP genes was responsive to cold treatment. Overexpressing TIP2;1, TIP3;2 or TIP4;3 markedly diminished the cold tolerance of maize seedlings, whereas loss-of-function mutants of TIP4;3 showed enhanced cold tolerance. Candidate gene-based association analysis indicated that a 328-bp transposon insertion in the TIP4;3 promoter was strongly associated with maize cold tolerance. The detailed regulatory mechanism will be presented.