Stomatal regulation

2.1. Anatomy: The Key to Movement

The secret of stomatal movement lies in the special structure of the guard cells (Hopkins & Hüner, 2009; Taiz et al., 2023).

1. Uneven thickening of the cell wall. In typical (kidney-shaped) guard cells, the wall facing the stomatal pore (inner) is significantly thicker than the wall in contact with neighboring cells (outer). In addition, the wall sections adjacent to the cell poles and the stomatal opening are also thickened. The inner wall often has a folded structure (Medvedev, 2012).

2. Orientation of cellulose microfibrils. This is perhaps the most important structural element. In most plant cells, cellulose microfibrils are arranged transversely, which restricts expansion in width. In guard cells, they are oriented radially, that is, like the spokes of a wheel, from the center to the periphery (Taiz et al., 2023; see also Connor et al., 2011). Thanks to this arrangement, when the cell increases in volume, its length grows less than its width, and it expands predominantly sideways. This causes the two guard cells to bend in an arc, moving away from each other, and the pore between them widens.

In grasses, guard cells are dumbbell-shaped: their ends are swollen, and the middle part is narrow. In them, microfibrils are oriented longitudinally, and when the swollen ends swell, the cells seem to push apart, opening a long pore (Taiz et al., 2023).

2.2. The Osmotic Pump: How Turgor is Created

Now that we understand the "mechanics" of movement, let's see what causes the guard cells to change their volume. The change in turgor is the result of active changes in the concentration of osmotically active substances inside the cells. The process of stomatal opening can be represented as a sequential chain of events, described in detail in several sources (Taiz et al., 2023; Hopkins & Hüner, 2009; Lambers & Oliveira, 2019; Medvedev, 2012).

1. Signal Reception. Under the influence of light (especially the blue spectrum) or changes in CO₂ concentration, transport proteins in the plasmalemma of the guard cells are activated. The main role is played by the proton pump (H⁺-ATPase)—an integral membrane protein that, expending ATP energy, pumps protons (H⁺) out of the cell (Schopfer & Brennicke, 2016; Taiz et al., 2023). This creates an electrochemical gradient: the inner side of the membrane becomes more negative, while protons accumulate outside.

2. Accumulation of Ions and Osmotica. The energy of this gradient is used for the entry of potassium ions (K⁺) into the cell. Through specialized K⁺ channels, K⁺ rushes into the guard cells. To balance the electrical charge, along with or following potassium, counter-ions—chloride anions (Cl⁻)—enter the cell, and, very importantly, malate anion (malate²⁻) is synthesized (Taiz et al., 2023; Lambers & Oliveira, 2019). This leads to a sharp increase in the concentration of ions in the vacuole and cytoplasm of the guard cells. The K⁺ concentration can increase from 50–100 mM to 400–800 mM (Taiz et al., 2023).

3. Water Influx. The intracellular environment becomes hypertonic relative to the surrounding tissues and the apoplast. The osmotic potential (Ψₛ) inside the guard cells becomes more negative (Medvedev, 2012). This creates a water potential gradient (Ψw) between the surrounding cells (with higher Ψw) and the guard cells (with lower Ψ_w). Water enters the guard cells osmotically, along this gradient. To facilitate and accelerate this process, specialized water channels—aquaporins—are inserted into the membrane (Lambers & Oliveira, 2019; Medvedev, 2012). For instance, plasma membrane aquaporins (PIP) and tonoplast aquaporins (TIP) play a key role here (Medvedev, 2012).

4. Volume Increase and Opening. The influx of water leads to an increase in cell volume and a rise in turgor pressure (Ψₚ). Due to the anisotropic structure of the cell wall described above, this pressure is realized not in uniform expansion, but in the bending of the cells, and the stomatal pore opens. Once the concentration of osmotica equilibrates or their influx decreases, the dynamic equilibrium shifts, and the process stops.

2.3. Closing—The Reverse Process

Stomatal closure is not a passive collapse but an active regulatory process. It is triggered when a signal of water deficit enters the cell (e.g., the hormone abscisic acid—ABA). In response to this signal:

1. Channels for the entry of K⁺ and Cl⁻ ions close.

2. Channels for the exit of these ions from the cell open (Taiz et al., 2023; Lambers & Oliveira, 2019).

3. Ions K⁺, Cl⁻, and malate leave the guard cells. The osmotic potential inside them increases (becomes less negative), water leaves the cells along the water potential gradient, turgor falls, and the pore closes.

Thus, the guard cells work like a "hydraulic piston" controlled by the concentration of salts and organic acids. This allows the plant to open and close stomata very quickly (within minutes), responding to changing environmental conditions and its own needs.

Main idea of the section: The plant does not control stomata directly, but indirectly—through changes in the concentration of solutes, which leads to water movement and, consequently, to changes in guard cell turgor. This elegant solution minimizes energy expenditure for movement by using the physical laws of osmosis.

Now that we know how this mechanism works, the next logical question is: who controls it? What external and internal signals cause the proton pump to turn on or off? In the next part of the lecture, we will examine which signals open the stomata and prepare the leaf for daytime work, and which, conversely, cause them to close, protecting the plant from dehydration.

Excellent, let's continue. In the previous section, we analyzed the "hydraulic mechanism" of the guard cells—exactly how changes in osmotic pressure lead to the opening or closing of the stomatal pore. Now let's move on to the most interesting part: what is the trigger for this mechanism? What signals cause the proton pumps to turn on, initiating the cascade of events leading to stomatal opening? It is important to emphasize right away: the plant is not a passive executor but an active regulator. It does not just "react" to signals but integrates information from the environment and its own metabolic state to make a balanced decision.

3.1. The Blue Light Signal: The Main "Alarm Clock"

The most powerful and rapid signal for stomatal opening is blue light. This signal is perceived by specialized photoreceptors—phototropins—located in the plasmalemma of guard cells (Taiz et al., 2023; Lambers & Oliveira, 2019; Hopkins & Hüner, 2009). Phototropins are proteins that are activated by blue light and trigger a complex signaling cascade.

1. Activation of the proton pump. Under the influence of phototropins, phosphorylation of H⁺-ATPase occurs, activating it. The pump begins to actively pump H⁺ out of the cell, creating an electrochemical gradient (Schopfer & Brennicke, 2016; Taiz et al., 2023).

2. Opening of K⁺ channels. This gradient is the driving force for the entry of K⁺ through specific channels that open upon membrane hyperpolarization.

3. Rapid ion accumulation. Following K⁺, Cl⁻ enters, and malate synthesis begins. As a result, the osmotic potential drops, water enters, and the stomata open.

Interestingly, the response to blue light is so rapid that it can be observed within 1–3 minutes after the start of illumination. Moreover, blue light is effective even at very low intensities, characteristic of morning twilight (Taiz et al., 2023). Therefore, it serves as the main signal for the stomata to "wake up" and prepare for daytime photosynthesis.

3.2. The Red Light Signal: Indirect Influence

Red light (especially in the 660-680 nm region) also stimulates stomatal opening, but its effect is mediated by photosynthesis. Chlorophyll in the mesophyll and possibly in the guard cells themselves absorbs red light, triggering photosynthesis. As a result:

1. Reduction of CO₂ in the intercellular spaces. Photosynthesis actively consumes CO₂, and its concentration in the leaf air spaces decreases (Lambers & Oliveira, 2019; Taiz et al., 2023).

2. Opening signal. The decrease in CO₂ concentration itself is a powerful signal for stomatal opening (see the next point). Thus, red light acts not directly but through changes in the metabolic state of the leaf.

3.3. The Signal of Decreased CO₂ Concentration: Metabolic Necessity

Stomata are sensitive to the concentration of carbon dioxide in the intercellular spaces (Cᵢ). When photosynthesis is active and Cᵢ drops below a certain threshold (usually around 200-250 μmol mol⁻¹ for C₃ plants), this is perceived by the guard cells as a signal of the need to open up to increase the influx of CO₂ (Taiz et al., 2023; Connor et al., 2011). This mechanism works even in the dark, but then the CO₂ level may rise due to respiration, which, conversely, causes closure. The mechanism of CO₂ perception is not fully understood, but it is known to involve changes in cell pH and ion channel activity (Lambers & Oliveira, 2019).

It is important to note that this works in the opposite direction as well: if the CO₂ concentration becomes too high (e.g., in a closed greenhouse or in still air), the stomata begin to close to limit the intake of carbon dioxide, which is already in excess. This demonstrates that the plant aims to maintain an optimal CO₂ concentration inside the leaf, rather than just opening as wide as possible.

3.4. Other Factors Promoting Opening

In addition to light and CO₂, stomatal opening can be stimulated by:

• High air humidity (reduced water vapor deficit). When the air is humid, the threat of dehydration is minimal, and stomata can open wider (Taiz et al., 2023).
• Optimal temperature. At excessively high or low temperatures, the processes of ATP synthesis and membrane pump operation are disrupted, and stomatal opening becomes less intense (Lambers & Oliveira, 2019).
• Hormonal status. For example, cytokinins, which are synthesized in the roots, can stimulate stomatal opening, signaling a favorable state of water supply (Medvedev, 2012).

Signal Integration: "Collective Decision"

The most amazing thing is that all these signals do not act individually. The guard cells constantly analyze a set of information: light intensity and quality, CO₂ level, air humidity, temperature, and even the availability of water in the soil (which we will discuss in the next section). This is like a complex processor that, based on multiple inputs, produces a single output signal—to open or close the stomata.

For example, in the morning, when blue light intensity is still low, but CO₂ has accumulated from nighttime respiration, the CO₂ signal may be dominant, causing partial opening. And when the sun rises higher, the blue light signal becomes stronger, and the stomata open fully. If heat and dry air simultaneously occur, the plant may decide to close the stomata, even despite the abundance of light.

Main idea of the section: The plant does not open its stomata for no reason. Opening is an active, energy-consuming action that is triggered only when a set of favorable signals is present, the main ones being blue light (as an indicator of the coming day) and a decrease in CO₂ concentration (as an indicator of photosynthetic demand). This "smart" decision allows the most efficient use of light for photosynthesis while avoiding excessive water loss when conditions are suboptimal.

Now that we know what makes the stomata open, we come to the next, equally important question: what makes them close? The plant must have "stop signals" that protect against dehydration. It turns out that the mechanisms of closure are no less sophisticated and include both a rapid response to water loss and "remote control" from the root system. This is exactly what we will discuss in the next section.

Excellent, let's continue our discussion on how the plant makes decisions. We learned that guard cells are the executive mechanism, and light and carbon dioxide are the main signals for opening. But the most amazing thing about this system is not the ability to open, but the ability to close in time. After all, if the plant loses water uncontrollably, it will die faster than from a lack of carbon dioxide. Therefore, stomata must have effective "brake" systems that activate when there is a threat of dehydration. These systems are surprisingly diverse and work at different levels—from rapid physical reactions to complex hormonal signals from the root.

4.1. Hydropassive Closure: Rapid Physical Protection

This is the simplest and fastest mechanism. If the plant loses water through transpiration faster than the roots can replenish it, the water potential of the leaves drops. This leads to:

1. Water loss by guard cells. Guard cells are not isolated from the overall water status of the leaf. If neighboring epidermal and mesophyll cells lose water and their water potential becomes lower, water begins to leave the guard cells osmotically (Taiz et al., 2023; Medvedev, 2012).

2. Turgor drop. A decrease in the volume of guard cells leads to a sharp drop in turgor pressure.

3. Passive pore closure. Since the structure of the cell wall is designed to maintain the pore open at high turgor, when turgor decreases, the cells lose their shape and close together. This is similar to how a deflating balloon becomes soft and loses its shape.

This mechanism is an emergency measure. It does not require energy expenditure and activates when the plant is already in a state of water deficit. It is a "safety valve" that prevents further water loss when the situation becomes critical. However, this method of protection has a drawback: by the time it activates, the plant is already suffering from dehydration.

4.2. Hydroactive (Hormonal) Closure: "Preemptive Strike" from the Root

A much "smarter" and more forward-looking way of protection is the active closure of stomata, initiated by a signal coming from the root system when the soil begins to dry out. This mechanism is described in many sources (Taiz et al., 2023; Lambers & Oliveira, 2019; Connor et al., 2011; Medvedev, 2012) and is one of the most striking examples of long-distance signaling in plants.

The key player here is the hormone abscisic acid (ABA).

The process looks like this:

1. Perception of soil dryness by roots. When the soil water potential decreases, the roots (especially their tips) begin to synthesize ABA. Interestingly, the signal for synthesis can be not only dehydration itself but also the mechanical compression of the soil as it dries. In addition, an increase in the pH of the xylem sap, which accompanies water deficit, also contributes to the accumulation of ABA in the leaves (Lambers & Oliveira, 2019).

2. Transport of the hormone to the leaves. ABA is loaded into the xylem stream and carried to the leaves with the transpiration flow. This is a very effective way of communication: the faster the transpiration, the faster the signal of water deficit reaches the leaf (Taiz et al., 2023).

3. Action of ABA on guard cells. Once in the leaf, ABA binds to specific receptors on the plasmalemma of the guard cells. This triggers a complex signaling cascade in which an increase in the concentration of calcium ions (Ca²⁺) in the cytosol and changes in pH play an important role (Lambers & Oliveira, 2019; Taiz et al., 2023).

4. Inactivation of the proton pump and opening of channels. Under the influence of ABA, H⁺-ATPase is inactivated, and the channels that release K⁺, Cl⁻, and malate ions from the cell open. This leads to a rapid efflux of osmotica, water outflow, a drop in turgor, and, consequently, stomatal closure.

What is the genius of this mechanism? The plant begins to close its stomata before critical leaf dehydration occurs! The signal from the root warns of impending danger. This allows the plant to "preemptively" reduce water consumption, maintaining the leaf water status at an acceptable level. This is called feed-forward regulation (Connor et al., 2011; Taiz et al., 2023).

Experiments brilliantly confirm this. If the roots of a plant are placed in soil that begins to dry out, and the plant itself is placed in a high-pressure chamber that compensates for the drop in leaf water pressure and artificially maintains its hydration, the stomata will still close. Because the signal has already arrived from the roots (Lambers & Oliveira, 2019). There are also known experiments with "split-root systems," where part of the roots is in wet soil and part in dry soil. In this case, the stomata partially close, even though on average the plant receives enough water. The signal from the drying roots dominates (Lambers & Oliveira, 2019).

4.3. The Signal of High CO₂ Concentration: Metabolic "Stop Command"

We have already mentioned that low CO₂ concentration stimulates stomatal opening. Naturally, high concentration acts in the opposite way. This is especially relevant at night, when photosynthesis does not occur, but respiration continues. As a result, CO₂ accumulates in the intercellular spaces, serving as a signal for stomatal closure to avoid unnecessary water loss (Taiz et al., 2023; Hopkins & Hüner, 2009).

Moreover, in dense plant stands or in closed greenhouses with poor ventilation, the CO₂ concentration can rise to levels that cause partial stomatal closure even during the day, reducing photosynthesis. This is important to consider in agronomic practice.

Signal Integration: Who Wins the Argument?

Thus, guard cells are under constant pressure from competing signals. On one hand, light and low CO₂ "shout": "Open!". On the other hand, ABA from the roots, high CO₂ concentration, and a drop in leaf turgor demand: "Close!". How does the plant resolve this conflict?

The answer depends on the strength of the signals. Water is a matter of life and death. Therefore, under drought conditions, the hormonal ABA signal often overrides the light signals. Stomata may remain closed even on a bright sunny day if the roots sense a lack of moisture. The plant "sacrifices" potential photosynthesis for survival. In physiology, this is known as a drought avoidance strategy (Connor et al., 2011).

Main idea of the section: Stomatal closure is not just cell "fatigue." It is the result of a complex "early warning" system that allows the plant to actively and preemptively save water, receiving signals from the root system, and to respond rapidly to changes in the metabolic status of the leaf, as well as to direct water loss. This makes stomatal regulation one of the most sophisticated and important regulatory mechanisms in the plant.

Now that we know which signals open and close the stomata, let's see how all of this works in dynamics—over the course of a typical day. How does the degree of stomatal opening change from dawn to dusk, and how do different types of plants (C₃, C₄, CAM) solve this problem differently? We will discuss this in the next, concluding section of our lecture.

Excellent, we have reached the concluding section of our lecture. We have analyzed the mechanism of stomatal operation, learned which signals open them and which close them. Now is the time to look at all of this in dynamics. After all, in nature, conditions are not static: the sun rises and sets, temperature and humidity change throughout the day, and the soil is either moistened by rain or dries out. How does the plant, this complex signal integrator, behave during the day? Let's trace a typical daily cycle of stomatal operation and see how the idea of compromise between water and carbon is realized in practice.

5.1. A Typical Diurnal Cycle: Morning — Day — Evening

For most mesophytic plants (typical temperate climate plants, cereal crops, legumes), the diurnal dynamics of stomatal conductance look approximately like this (Connor et al., 2011; Taiz et al., 2023; Lambers & Oliveira, 2019; Medvedev, 2012):

1. Pre-dawn period: Stomata are closed. At night, photosynthesis does not occur, but respiration continues, so CO₂ accumulates in the leaf intercellular spaces. High CO₂ concentration is a signal for closure (see section 4.3). In addition, the main signal for opening—blue light—is absent. Thus, stomata are closed to avoid useless water loss.

2. Dawn (opening phase): With the first rays of the sun, processes leading to opening are initiated:

• Blue light appears, which, through phototropins, activates proton pumps and initiates the accumulation of K⁺ in the guard cells. This is the "main alarm clock" (Taiz et al., 2023).
• Photosynthesis is activated. Since CO₂ accumulated overnight, its concentration in the leaf begins to drop rapidly. The decrease in Cᵢ is a powerful metabolic signal for opening (Lambers & Oliveira, 2019).
• Temperature and air humidity in the morning are usually moderate, which does not create acute water stress, and the "brake" signals (ABA, hydropassive closure) are weak.

As a result, the stomata open rapidly, reaching maximum conductance in the first half of the day. This allows the plant to make maximum use of the morning hours for photosynthesis, when there is still enough moisture in the soil and it is not too hot.

3. Midday (noon depression or "lunch break"): This is the most interesting and telling moment. On a hot, sunny day, when transpiration is maximal, stomata may begin to partially close, even despite the abundance of light and ongoing photosynthesis. This phenomenon is called noon depression of transpiration or midday stomatal closure (Lambers & Oliveira, 2019; Connor et al., 2011; Hopkins & Hüner, 2009).

Why does this happen? Because by noon, the action of closing signals intensifies:

• High temperature and air humidity deficit: The air becomes drier, the water vapor gradient between the leaf and the atmosphere increases, which accelerates water loss. This alone can cause hydropassive closure.
• ABA activation: If the roots begin to sense soil drying, the flow of ABA to the leaves increases, causing hydroactive closure (Taiz et al., 2023).
• Accumulation of photoassimilates: Intense photosynthesis can lead to the accumulation of sugars in the leaf, which is also a feedback signal for reducing stomatal conductance (Lambers & Oliveira, 2019).

As a result, stomatal conductance can drop by 30-50% from the morning maximum. This is a protective mechanism: the plant consciously sacrifices part of its potential photosynthesis to avoid critical dehydration and damage to the photosynthetic apparatus. This is "the compromise in action."

4. Evening (closing phase): Toward sunset, light intensity decreases, and photosynthesis slows down. The CO₂ accumulated during the day is no longer actively absorbed, and its concentration in the intercellular spaces begins to rise. Opening signals weaken, while closing signals (high CO₂, possible ABA accumulation) begin to dominate. Stomata gradually close, and by night they are completely shut to avoid water loss in the dark.

5.2. Influence of Environmental Conditions on Diurnal Dynamics

It is important to understand that the "ideal" cycle described above is only a scheme. In practice, it varies greatly depending on environmental conditions and the type of plant.

• Soil moisture: If the soil is well moistened, the noon depression is mild or absent. If the soil is dry, the peak of opening may shift to earlier morning, and the decline itself may be deeper and more prolonged (Connor et al., 2011; Taiz et al., 2023).
• Air humidity: In dry air, stomata may close earlier and more strongly than in humid air. In the tropics, where humidity is high, stomata may remain open almost all day.
• Temperature: At extremely high temperatures (above 35-40 °C), the activity of enzymes, including the proton pump, decreases, which can lead to spontaneous stomatal closure (Lambers & Oliveira, 2019).
• Atmospheric CO₂ concentration: Under elevated CO₂ conditions (e.g., in greenhouses or under global climate change), stomata open less, which increases the plant's water use efficiency (Lambers & Oliveira, 2019; Taiz et al., 2023).

5.3. Features of Diurnal Dynamics in Plants with Different Photosynthesis Types (C₃, C₄, CAM)

Here we touch only on behavioral differences, without delving into the biochemistry of photosynthesis, which will be discussed in detail in the next module.

C₃ plants (wheat, rice, soybean, potato): They have the most pronounced noon depression. They are very sensitive to water deficit and often show two peaks of photosynthesis—morning and evening—separated by a "lunch decline" (Connor et al., 2011; Lambers & Oliveira, 2019). Their stomatal conductance is strongly correlated with light intensity but is easily suppressed by ABA and water stress.

C₄ plants (corn, sugarcane, sorghum): They have higher water use efficiency. Their stomata may remain sufficiently open in the middle of the day even at low humidity, as they are able to maintain a lower CO₂ concentration in the leaf. The noon depression in them is less pronounced (Taiz et al., 2023; Medvedev, 2012). However, under severe drought, they too close their stomata.

CAM plants (cacti, aloe, agaves, crassulaceae): This is the most radical way to solve the "water-carbon" compromise. In these plants, stomata open at night and are closed during the day. This is because their photosynthesis is separated in time: at night, CO₂ is fixed and stored as organic acids (malate), and during the day, this CO₂ is released and used in photosynthetic reactions with closed stomata (Taiz et al., 2023; Lambers & Oliveira, 2019; Medvedev, 2012; Hopkins & Hüner, 2009).

So, if in C₃ and C₄ plants the stomata are open during the day to capture CO₂ directly from the air, in CAM plants—they are open at night, when temperatures are lower and water loss is minimal. This allows CAM plants to survive in the most arid conditions. Their transpiration coefficient is the lowest—30-150 (Medvedev, 2012).

5.4. Long-Term Adaptations: "Tuning" the System

It is important to add that the plant can adapt not only during the day but also over a longer time frame. For example, during prolonged drought, plants can:

• Reduce the number of stomata per unit leaf area (Medvedev, 2012).
• Increase the thickness of the cuticle, reducing cuticular transpiration (Medvedev, 2012; Taiz et al., 2023).
• Change leaf size and shape, reducing the evaporating surface (Connor et al., 2011; Lambers & Oliveira, 2019).
• Develop a more powerful root system for better water supply (Lambers & Oliveira, 2019).

Main idea of the section: The diurnal dynamics of stomata is not just a mechanical response to light but a complex, dynamic process in which the plant constantly balances between the need for photosynthesis and the risk of dehydration. This dynamics can vary greatly depending on environmental conditions and the ecological strategy of the species. From "classic" C₃ plants, sensitive to drought, to "extremophile" CAM plants, which minimize water loss at the cost of slowed growth. This demonstrates the amazing plasticity and adaptive capabilities of the plant world, which underlie our agronomic practice.

With this, we conclude the discussion of stomatal regulation. In the next lecture, we will move up to the level of the whole plant and see how the coordinated work of roots, xylem, and leaves creates a unified water balance that determines the productivity and resilience of agroecosystems.

We have reached the conclusion of our lecture. We started with a fundamental question: how does the plant resolve the main compromise of its life—the need to obtain carbon dioxide for photosynthesis while not dying from dehydration? We traversed the path from understanding this global problem to its solution at the cellular level, studied the complex signaling network, and saw how it all works in the dynamics of a real day. It is time to summarize and consolidate the main conclusions that will form the basis for understanding subsequent topics.

Bridge to the Next Lecture

We have studied in detail the work of an individual stoma—a microscopic valve regulating gas exchange and water loss at the leaf level. Now we need to take an important step upward—from the cellular and organ levels to the whole organism.

In the next lecture, which will be devoted to the water balance of the plant, we will consider how the work of individual stomata is coordinated with the activity of the entire system: how roots absorb water, how xylem transports it to the leaves, how leaves distribute water between transpiration and photosynthesis, and how the plant as a whole maintains its hydration in constantly changing environmental conditions. We will see that stomata are only the tip of the iceberg, and to understand the water regime of a crop, it is necessary to consider the entire "soil – plant – atmosphere" system. This will be our next topic.

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