Overexpression of tonoplast Ca2+-ATPase in guard cells synergistically enhances stomatal opening and drought tolerance.

Stomata play a crucial role in plants by controlling water status and responding to drought stress. However, simultaneously improving stomatal opening and drought tolerance has proven to be a significant challenge. To address this issue, we employed the OnGuard quantitative model, which accurately represents the mechanics and coordination of ion transporters in guard cells. With the guidance of OnGuard, we successfully engineered plants that overexpressed the main tonoplast Ca2+-ATPase gene, ACA11, which promotes stomatal opening and enhances plant growth. Surprisingly, these transgenic plants also exhibited improved drought tolerance due to reduced water loss through their stomata. Again, OnGuard assisted us in understanding the mechanism behind the unexpected stomatal behaviors observed in the ACA11 overexpressing plants. Our study revealed that the overexpression of ACA11 facilitated the accumulation of Ca2+ in the vacuole, thereby influencing Ca2+ storage and leading to an enhanced Ca2+ elevation in response to abscisic acid. This regulatory cascade finely tunes stomatal responses, ultimately leading to enhanced drought tolerance. Our findings underscore the importance of tonoplast Ca2+-ATPase in manipulating stomatal behavior and improving drought tolerance. Furthermore, these results highlight the diverse functions of tonoplast-localized ACA11 in response to different conditions, emphasizing its potential for future applications in plant enhancement.

Surrounded by luxury: The necessities of subsidiary cells.

The evolution of stomata marks one of the key advances that enabled plants to colonise dry land while allowing gas exchange for photosynthesis. In large measure, stomata retain a common design across species that incorporates paired guard cells with little variation in structure. By contrast, the cells of the stomatal complex immediately surrounding the guard cells vary widely in shape, size and count. Their origins in development are similarly diverse. Thus, the surrounding cells are likely a luxury that the necessity of stomatal control cannot do without (with apologies to Oscar Wilde). Surrounding cells are thought to support stomatal movements as solute reservoirs and to shape stomatal kinetics through backpressure on the guard cells. Their variety may also reflect a substantial diversity in function. Certainly modelling, kinetic analysis and the few electrophysiological studies to date give hints of much more complex contributions in stomatal physiology. Even so, our knowledge of the cells surrounding the guard cells in the stomatal complex is far from complete.

Arabidopsis SNARE SYP132 impacts on PIP2;1 trafficking and function in salinity stress.

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.

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.

Mother trees, altruistic fungi, and the perils of plant personification.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Speedy stomata of a C4 plant correlate with enhanced K+ channel gating.

Stomata are microscopic pores at the surface of plant leaves that facilitate gaseous diffusion to support photosynthesis. The guard cells around each stoma regulate the pore aperture. Plants that carry out C4 photosynthesis are usually more resilient than C3 plants to stress, and their stomata operate over a lower dynamic range of CO2 within the leaf. What makes guard cells of C4 plants more responsive than those of C3 plants? We used gas exchange and electrophysiology, comparing stomatal kinetics of the C4 plant Gynandropsis gynandra and the phylogenetically related C3 plant Arabidopsis thaliana. We found, with varying CO2 and light, that Gynandropsis showed faster changes in stomata conductance and greater water use efficiency when compared with Arabidopsis. Electrophysiological analysis of the dominant K+ channels showed that the outward-rectifying channels, responsible for K+ loss during stomatal closing, were characterised by a greater maximum conductance and substantial negative shift in the voltage dependence of gating, indicating a reduced inhibition by extracellular K+ and enhanced capacity for K+ flux. These differences correlated with the accelerated stomata kinetics of Gynandropsis, suggesting that subtle changes in the biophysical properties of a key transporter may prove a target for future efforts to engineer C4 stomatal kinetics.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

OnGuard3e: A predictive, ecophysiology-ready tool for gas exchange and photosynthesis research.

Gas exchange across the stomatal pores of leaves is a focal point in studies of plant-environmental relations. Stomata regulate atmospheric exchange with the inner air spaces of the leaf. They open to allow CO2 entry for photosynthesis and close to minimize water loss. Models that focus on the phenomenology of stomatal conductance generally omit the mechanics of the guard cells that regulate the pore aperture. The OnGuard platform fills this gap and offers a truly mechanistic approach with which to analyse stomatal gas exchange, whole-plant carbon assimilation and water-use efficiency. Previously, OnGuard required specialist knowledge of membrane transport, signalling and metabolism. Here we introduce OnGuard3e, a software package accessible to ecophysiologists and membrane biologists alike. We provide a brief guide to its use and illustrate how the package can be applied to explore and analyse stomatal conductance, assimilation and water use efficiencies, addressing a range of experimental questions with truly predictive outputs.

Analyzing Protein-Protein Interactions Using the Split-Ubiquitin System.

The split-ubiquitin technology was developed over 20 years ago as an alternative to Gal4-based, yeast-two-hybrid methods to identify interacting protein partners. With the introduction of mating-based methods for split-ubiquitin screens, the approach has gained broad popularity because of its exceptionally high transformation efficiency, utility in working with full-length membrane proteins, and positive selection with little interference from spurious interactions. Recent advances now extend these split-ubiquitin methods to the analysis of interactions between otherwise soluble proteins and tripartite protein interactions.

Engineering stomata for enhanced carbon capture and water-use efficiency.

Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. As gatekeepers that balance CO2 entry for photosynthesis against transpirational water loss, they are a focal point for efforts to improve crop performance, especially in the efficiency of water use, within the changing global environment. Until recently, engineering strategies had focused on stomatal conductance in the steady state. These strategies are limited by the physical constraints of CO2 and water exchange such that gains in water-use efficiency (WUE) commonly come at a cost in carbon assimilation. Attention to stomatal speed and responsiveness circumvents these constraints and offers alternatives to enhancing WUE that also promise increases in carbon assimilation in the field.

Environmental morphing enables informed dispersal of the dandelion diaspore.

Animal migration is highly sensitised to environmental cues, but plant dispersal is considered largely passive. The common dandelion, Taraxacum officinale, bears an intricate haired pappus facilitating flight. The pappus enables the formation of a separated vortex ring during flight; however, the pappus structure is not static but reversibly changes shape by closing in response to moisture. We hypothesised that this leads to changed dispersal properties in response to environmental conditions. Using wind tunnel experiments for flow visualisation, particle image velocimetry, and flight tests, we characterised the fluid mechanics effects of the pappus morphing. We also modelled dispersal to understand the impact of pappus morphing on diaspore distribution. Pappus morphing dramatically alters the fluid mechanics of diaspore flight. We found that when the pappus closes in moist conditions, the drag coefficient decreases and thus the falling velocity is greatly increased. Detachment of diaspores from the parent plant also substantially decreases. The change in detachment when the pappus closes increases dispersal distances by reducing diaspore release when wind speeds are low. We propose that moisture-dependent pappus-morphing is a form of informed dispersal allowing rapid responses to changing conditions.

Engineering a K+ channel 'sensory antenna' enhances stomatal kinetics, water use efficiency and photosynthesis.

Stomata of plant leaves open to enable CO2 entry for photosynthesis and close to reduce water loss via transpiration. Compared with photosynthesis, stomata respond slowly to fluctuating light, reducing assimilation and water use efficiency. Efficiency gains are possible without a cost to photosynthesis if stomatal kinetics can be accelerated. Here we show that clustering of the GORK channel, which mediates K+ efflux for stomatal closure in the model plant Arabidopsis, arises from binding between the channel voltage sensors, creating an extended 'sensory antenna' for channel gating. Mutants altered in clustering affect channel gating to facilitate K+ flux, accelerate stomatal movements and reduce water use without a loss in biomass. Our findings identify the mechanism coupling channel clustering with gating, and they demonstrate the potential for engineering of ion channels native to the guard cell to enhance stomatal kinetics and improve water use efficiency without a cost in carbon fixation.

Unidirectional versus bidirectional brushing: Simulating wind influence on Arabidopsis thaliana.

Plants acclimate to various types of mechanical stresses through thigmomorphogenesis and alterations in their mechanical properties. Although resemblance between wind- and touch-induced responses provides the foundation for studies where wind influence was mimicked by mechanical perturbations, factorial experiments revealed that it is not always straightforward to extrapolate results induced by one type of perturbation to the other. To investigate whether wind-induced changes in morphological and biomechanical traits can be reproduced, we subjected Arabidopsis thaliana to two vectorial brushing treatments. Both treatments significantly affected the length, mechanical properties and anatomical tissue composition of the primary inflorescence stem. While some of the morphological changes were found to be in line with those induced by wind, changes in the mechanical properties exhibited opposite trends irrespective of the brushing direction. Overall, a careful design of the brushing treatment gives the possibility to obtain a closer match to wind-induced changes, including a positive tropic response.

What can mechanistic models tell us about guard cells, photosynthesis, and water use efficiency?

Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. The pores open to enable CO2 entry for photosynthesis and close to reduce transpirational water loss. How stomata respond to the environment has long attracted interest in modeling as a tool to understand the consequences for the plant and for the ecosystem. Models that focus on stomatal conductance for gas exchange make intuitive sense, but such models need also to connect with the mechanics of the guard cells that regulate pore aperture if we are to understand the 'decisions made' by stomata, their impacts on the plant and on the global environment.