Stress perception

1.1. Stress as a Disruption of Homeostasis

Let's recall the basic definition of stress from the previous lecture. Life is the maintenance of an ordered state, far from equilibrium with the environment (Hopkins and Hüner, 2009). This state is called homeostasis. Any change in the external environment that disrupts this homeostasis is stress. But how does a plant know about such a disruption?

It is crucial to understand the key idea here: the primary signal is always a physical or physicochemical change within the cell that occurs before the plant "realizes" there is a problem and begins to respond.

Simply put: the plant hasn't "decided" anything yet—it hasn't turned on its defense genes or started synthesizing stress proteins. But the physical properties of its cells have already changed. These changes are the primary signals.

1.2. The Cell as a Physical System

To understand the nature of primary signals, we must view the cell not only as a biochemical system but also as a physical system. The cell has:

• Membranes—a lipid bilayer with embedded proteins, possessing specific fluidity and permeability.
• Cell wall—a rigid yet elastic structure that experiences mechanical tension.
• Cytoplasm—an aqueous solution containing dissolved ions, metabolites, and macromolecules.
• Vacuole—a large compartment generating osmotic pressure (turgor).

All these structures have physical characteristics that can change when external conditions change. These changes become the first signals for the plant.

1.3. Examples of Primary Signals

Let's look at several specific examples of what can serve as a primary signal.

1.4. Systems for Perceiving Primary Signals

How does the cell perceive these primary signals? Special mechanisms exist for this:

1. Physical perception—mechanical changes in the membrane or cell wall that lead to the opening of mechanosensitive ion channels (Taiz et al., 2023).

2. Biophysical perception—changes in the conformation or activity of proteins in response to changes in temperature, pH, or ionic strength (Taiz et al., 2023).

3. Metabolic perception—accumulation of toxic or signaling metabolites (ROS, methylglyoxal, etc.) (Taiz et al., 2023).

4. Biochemical perception—interaction of a signaling molecule (e.g., hormone, ion) with a specialized receptor (Taiz et al., 2023).

5. Epigenetic perception—changes in chromatin structure in response to stress (Taiz et al., 2023).

1.5. From Primary Signal to Secondary Signal

So, we've clarified what a primary signal is. It is always a physical or physicochemical change in the cell that occurs before the active response begins. This is not the plant's decision, but merely a "message" that something has changed.

However, the primary signal must be translated into a language the cell understands—a biochemical signal. Such a signal is called a secondary signal or signal messenger.

The most common secondary signals in plant cells are:

• Calcium ions (Ca²⁺)—a fast and universal signal,
• Reactive oxygen species (ROS),
• Cyclic ADP-ribose,
• Phosphatidic acid and other lipid signals.

We will discuss the mechanisms of translating primary signals into secondary ones and the subsequent signaling cascades in the following lectures.

Interim Summary

1. The primary signal is always a physical or physicochemical change in the cell that occurs before the plant begins to actively respond to stress.

2. Temperature is sensed by the membrane—through changes in its fluidity, which are perceived by membrane proteins.

3. Water deficit is sensed through changes in turgor—reduced pressure on the cell wall and altered membrane tension (not via ABA or stomatal closure).

4. Changes in ionic composition are sensed directly—ions interact with membrane proteins.

5. Oxygen deficiency is sensed through changes in redox state—the ratio of reduced to oxidized electron carriers.

6. The oxidative burst occurs as one of the first responses to stress because ROS are constantly produced in the cell, and stress disrupts the balance between their production and scavenging.

In the following chapters, we will examine how these primary signals are converted into secondary messengers, activate signaling pathways, change gene expression, and ultimately lead to the formation of a plant's defense response.

2.1. Water Potential and Its Components

To understand how a plant perceives water deficit, we must recall what water potential (Ψ_w) is—a thermodynamic measure of the free energy of water in a system. Water potential determines the direction of water movement: it always moves from an area of higher (less negative) potential to an area of lower (more negative) potential (Lambers and Oliveira, 2019). In a plant cell, water potential is the sum of several components (Медведев, 2012; Lambers and Oliveira, 2019):

where:

• Ψ_s — osmotic potential (related to the concentration of solutes, always negative);
• Ψ_p — pressure potential (or hydrostatic pressure, positive in a turgid cell, created by turgor pressure);
• Ψ_g — gravitational potential (important for tall trees, usually negligible for herbaceous plants);
• Ψ_m — matrix potential (related to capillary forces in the cell wall and soil, always negative).

In a typical plant cell with adequate water supply, the pressure potential (Ψp) is a positive value (0.5–1.5 MPa) and balances the negative osmotic potential (Ψs), so that the total water potential is near zero or slightly negative. This positive Ψ_p is what we call turgor pressure—the force with which the cell contents press against the cell wall, providing tissue rigidity (Медведев, 2012).

2.2. Decreased Turgor—The Primary Signal for Water Deficit

When a plant encounters water deficit conditions (dry soil, high transpiration), the roots cannot compensate for water loss. Water leaves the cells, primarily from the vacuole, across membranes following the water potential gradient. The protoplast volume decreases, and the pressure on the cell wall drops. This leads to a reduction in Ψ_p—the pressure potential. Turgor is lost (Schopfer and Brennicke, 2016).

It is precisely this decrease in turgor pressure that is the earliest and most direct physical consequence of water deficit. This change occurs before abscisic acid (ABA) is synthesized or stomata close. The decrease in turgor is the primary signal (Taiz et al., 2023).

How does the cell "sense" this pressure decrease? It is crucial to understand: the cell does not have specialized pressure receptors in the conventional sense. Instead, the decrease in turgor changes the mechanical tension (stress) of the cell wall and the plasmalemma. The membrane, which in a turgid state is tightly pressed against the cell wall and under tension, slackens upon water loss, develops folds, and its curvature and mechanical state change (Медведев, 2012; Taiz et al., 2023). This is no longer a biochemical change but a purely physical change, which becomes accessible for perception by proteins embedded in the membrane.

2.3. Cell Mechanosensitivity: Sensing Membrane Tension

How can the cell "read" the change in membrane mechanical tension? Plant cells possess mechanosensitive ion channels (Taiz et al., 2023). These channels are transmembrane proteins that change their conformation and open or close in response to mechanical deformation of the lipid bilayer. When turgor decreases and membrane tension weakens, some of these channels alter their activity.

This primarily concerns calcium channels (e.g., the mechanosensitive channel MSL (MscS-like) and the family of channels associated with hyperosmotic stress). The decrease in turgor can lead to the opening of calcium channels and the influx of Ca²⁺ ions from the apoplast (or from intracellular stores) into the cytosol (Taiz et al., 2023). An increase in cytosolic calcium concentration is a classic secondary signal that triggers a whole cascade of events: activation of calcium-dependent protein kinases, protein phosphorylation, changes in gene expression. However, it's important to emphasize that the calcium influx itself is already a response to the primary signal, i.e., to the change in tension.

Besides calcium, changes in membrane tension can affect the activity of other ion channels (e.g., potassium, chloride) and even the activity of aquaporins—water channels that can also be regulated by mechanical tension (Lambers and Oliveira, 2019). Thus, a physical change is transformed into electrical and ionic signals that the cell can understand.

2.4. From Physical Signal to Biochemical Signal: The Role of ABA

In the early stages of water deficit, other signaling systems are activated in the cells besides calcium. One of the fastest responses is increased production of reactive oxygen species (ROS) in the apoplast, often associated with the activation of NADPH oxidases (RBOH) (Taiz et al., 2023). ROS, in turn, also act as signaling molecules; they can modify proteins and influence ion channels.

Only at later stages, when water deficit becomes prolonged, does the synthesis of the hormone abscisic acid (ABA) begin in the cells. ABA synthesis is induced precisely by the signals arising from turgor loss (Schopfer and Brennicke, 2016). ABA is a hormonal signal that is transported throughout the plant, particularly to the leaves, where it causes stomatal closure (reducing water loss) and metabolic reprogramming. However, it is important to remember: ABA is not the primary signal, but a secondary response, triggered after the cells have already "felt" water loss through the decrease in turgor.

Thus, the sequence of events is as follows:

1. Decrease in soil water potential → water efflux from root and leaf cells.

2. Decrease in turgor pressure (Ψ_p) → change in mechanical tension of the membrane and cell wall (primary signal).

3. Activation of mechanosensitive channels → Ca²⁺ influx and changes in ion fluxes (secondary signals).

4. Activation of protein kinases, NADPH oxidases → ROS production, protein phosphorylation (early biochemical reactions).

5. Induction of ABA synthesis (hormonal signal) and initiation of long-term adaptive programs (stomatal closure, synthesis of osmoprotectants, changes in gene expression).

2.5. Root Signals: How the Root Reports Soil Dryness

The perception of water deficit by roots is of particular interest because the root is the first to contact drying soil. Even if the leaves still maintain a relatively high water potential, the roots can sense soil dryness and initiate a signaling pathway transmitted to the shoot (Taiz et al., 2023). In this case, ABA often serves as the signaling substance, synthesized in the roots and transported via the xylem to the leaves, causing stomatal closure. This mechanism allows the plant to respond proactively to approaching drought, without waiting for dehydration to reach a critical level. However, even in this case, the signal for ABA synthesis in the roots is precisely the decrease in turgor in root cells—again a physical change (Schopfer and Brennicke, 2016).

2.6. Why is Growth More Sensitive to Water Deficit Than Photosynthesis?

Interestingly, different physiological processes have different sensitivities to decreases in water potential. For example, cell expansion growth (stem and root elongation) is inhibited even with a slight decrease in turgor, whereas photosynthesis suffers later (Paulsen, 1994; Медведев, 2012). This is easily explained because expansion growth directly depends on turgor pressure: cell enlargement requires that intracellular pressure exceeds a threshold value necessary for cell wall deformation. Loss of turgor immediately affects growth rate (Schopfer and Brennicke, 2016). Photosynthesis is mainly disrupted due to stomatal closure (which occurs as a protective response mediated by ABA) and the direct effects of dehydration on enzymes and chloroplast structure. This difference in sensitivity further confirms that perception of water deficit begins at the physical level of the cell, not at the biochemical level of the whole organ.

Thus, the answer to the question "how does a plant sense water deficit?" can be summarized: through changes in the mechanical tension of membranes and cell walls, caused by a drop in turgor pressure, which is then transformed into a chain of chemical signals, including Ca²⁺ ions, ROS, and ultimately the hormone ABA.

3.1. The Membrane as a Physical Structure Sensitive to Temperature

Cell membranes are lipid bilayers—two-dimensional fluids in which phospholipid molecules and proteins possess a certain mobility. This state is called the liquid-crystalline or fluid state. Like any liquid, this bilayer has characteristics that depend on temperature: fluidity and viscosity (Schopfer and Brennicke, 2016; Lambers and Oliveira, 2019).

When temperature rises, lipid molecules gain additional kinetic energy. They move faster, rotate, and change their position within the plane of the membrane. This leads to an increase in membrane fluidity. The degree of fluidity is determined by many factors, including the length and degree of unsaturation of fatty acid tails: the more unsaturated bonds (kinks in the chains), the more fluid the membrane remains at lower temperatures (Paulsen, 1994; Schopfer and Brennicke, 2016).

Upon cooling, conversely, the kinetic energy of molecules decreases, and the membrane becomes more viscous, rigid, and less permeable. At a certain critical temperature, the lipid bilayer can undergo a phase transition from the liquid-crystalline state to a more ordered solid-gel state, similar to how vegetable oil solidifies in a refrigerator (Taiz et al., 2023). Such a transition dramatically changes the membrane's physical properties—its permeability, the activity of embedded proteins, and its ability to maintain ion gradients. These physical changes are the primary temperature signal for the cell (Lambers and Oliveira, 2019).

3.2. Why the Membrane, and Not Something Else?

Why is temperature sensed specifically by the membrane, rather than, say, the cytoplasm or cell wall? There are several reasons:

1. The membrane is an ordered structure. Unlike the cytoplasm, which is an aqueous solution without long-range order, the membrane is a highly organized structure. A change in temperature leads to a change in the degree of order of this structure, which is easily detectable.

2. The membrane is at a phase interface. The membrane is the interface between two aqueous compartments (cytoplasm and apoplast, or matrix and intermembrane space). The physical properties of this interface strongly depend on temperature.

3. Membrane proteins are functionally dependent on the lipid environment. The function of membrane proteins (transporters, channels, receptors) depends on the fluidity of the surrounding lipids. Changes in fluidity lead to changes in their conformation and, consequently, activity.

4. Temperature receptors are embedded in the membrane. Even if specific protein thermosensors exist, they are immersed in the membrane and "read" its state, rather than sensing temperature directly (Taiz et al., 2023; Lambers and Oliveira, 2019).

3.3. How Does the Membrane "Transmit" Temperature Information?

The change in the physical properties of the membrane must be translated into the language of biochemical signals. How does this happen?

1. Activation of ion channels. Changes in membrane fluidity and thickness can induce conformational changes in embedded ion channels. For example, some calcium channels are sensitive to mechanical tension in the membrane, which in turn depends on temperature. Heat or cold exposure can open these channels, causing an influx of Ca²⁺ into the cytosol (Taiz et al., 2023).

2. Changes in enzyme activity. Enzymes embedded in the membrane (e.g., NADPH oxidases, phospholipases) can change their activity when the physical state of the lipid bilayer changes. This leads to the formation of secondary messengers—for instance, activation of phospholipase C can lead to the formation of inositol triphosphate (IP₃), which releases calcium from intracellular stores (Lambers and Oliveira, 2019).

3. Activation of receptor proteins. Some membrane proteins function as thermosensors. For example, the protein COLD1 (Chilling Tolerance Divergence 1), identified in rice, is a membrane protein involved in cold perception. It is thought to interact with a G-protein and alter its activity upon temperature decrease, leading to activation of calcium signaling (Ma et al., 2015, cited in Taiz et al., 2023). Another example is histidine kinases in cyanobacteria, which are classic two-component sensory systems also embedded in the membrane (Murata and Los, 1997, cited in Taiz et al., 2023). Thus, even specialized sensors are "tuned" to perceive the state of the membrane.

4. Changes in membrane permeability. The phase transition of the membrane to the gel state (during severe cooling) can sharply increase its permeability, especially for ions and small metabolites. Ion leakage from the cell—a classic symptom of chilling injury—is a consequence of this physical change, not of biochemical dysfunction (Schopfer and Brennicke, 2016).

3.4. Temperature and Heat Shock Protein Synthesis

A special mention deserves the famous heat shock response (synthesis of heat shock proteins, HSPs). When the temperature rises above a certain threshold, cells activate the synthesis of a special class of proteins that function as chaperones—they help other proteins fold correctly and prevent their aggregation during denaturation (Schopfer and Brennicke, 2016; Lambers and Oliveira, 2019).

Induction of HSP synthesis is associated with the activation of heat shock factors (HSF). Under normal conditions, HSFs are in an inactive state, bound to Hsp70/Hsp90 proteins. At elevated temperatures, these chaperone proteins switch to binding denatured proteins, releasing HSFs. Free HSFs trimerize, enter the nucleus, and activate transcription of HSP genes (Nakamoto and Akter, 2020; Taiz et al., 2023). It is important to note that this process is triggered precisely by protein damage, not by a direct effect of temperature on the membrane.

However, in the case of HSPs and other high-temperature responses, the membrane still plays an important role. Firstly, the change in membrane fluidity may be the initial signal leading to the activation of calcium channels and other pathways that can interact with the regulation of HSP gene expression. Secondly, HSP synthesis itself can protect the membrane: some small heat shock proteins (sHSPs) can bind to the membrane and stabilize it, preventing phase transition and ion leakage (Nakamoto and Akter, 2020).

3.5. Cold Shock and Hardening

A similar picture is observed when temperature decreases: changes in membrane fluidity lead to the activation of signaling pathways characteristic of cold stress. One of the key pathways is the CBF/DREB1 cascade in Arabidopsis and other temperate-climate plants (Taiz et al., 2023). Induction of this pathway begins with the perception of membrane changes (possibly through the mechanisms described above) and leads to the activation of CBF transcription factors, which in turn activate the expression of numerous cold-regulated genes (COR genes).

An important aspect of temperature perception is hardening (acclimation)—the ability of plants to increase their tolerance to cold or heat after prior exposure to sublethal temperatures (Schopfer and Brennicke, 2016; Paulsen, 1994). Hardening involves changes in membrane lipid composition (e.g., increasing the proportion of unsaturated fatty acids during cold hardening), which raises the temperature of the membrane gel-phase transition. This essentially changes the physical structure of the "sensor" itself, making the membrane less sensitive to cold stress. This is one of the most compelling examples of how fundamentally the physical properties of membranes determine temperature perception in plants.

Thus, the answer to "why is temperature sensed by the membrane?" is: because the membrane is a physical structure whose properties (fluidity, viscosity, phase state) are directly and predictably dependent on temperature. Changes in these properties alter the function of membrane-embedded proteins (channels, enzymes, receptors), generating intracellular signals (calcium, redox signals, activation of protein kinases) that subsequently trigger specific responses to heat or cold. In this sense, the membrane acts as a thermosensor—not as a specialized sense organ, but as a physically temperature-sensitive structure that transduces a physical stimulus into a biochemical signal.

4.1. Ions as Physical and Chemical Agents

When the concentration of ions in the soil solution changes, it affects the plant in at least two ways.

First, the osmotic effect: any dissolved substance lowers the water potential of the solution (Lambers and Oliveira, 2019). An increase in salt concentration (e.g., NaCl) in the soil lowers the water potential of the external solution. Since water moves from an area of higher potential to an area of lower potential, water begins to leave the root cells. This leads to turgor loss—exactly the same primary signal we discussed in the previous section (see Part 2). Thus, salt stress has an osmotic component, and it is perceived through mechanisms already familiar to us: decreased turgor pressure, changes in membrane tension, activation of mechanosensitive channels (Taiz et al., 2023).

However, unlike purely water deficit (drought), salt stress also has an ionic effect. Salt ions (primarily Na⁺ and Cl⁻) can penetrate the cell and directly affect its metabolism. Here, they act not only as toxic substances but also as signaling molecules. Even ions that are not toxic at normal concentrations (e.g., K⁺) can serve as signals when their concentration changes.

4.2. How Does the Cell "Sense" Changes in Ion Concentration?

Unlike turgor pressure or temperature, changes in ionic composition can be perceived in several ways.

4.3. Specifics of Salt Stress Perception

Salt stress (high NaCl content) is a classic example of how a change in environmental ionic composition is perceived by the cell. In plants, Na⁺ is generally toxic at high concentrations because it competes with K⁺ for binding to enzymes and transporters (Paulsen, 1994; Taiz et al., 2023). However, Na⁺ does not simply enter the cell passively—its uptake is regulated, and the cell can "sense" it even before the concentration becomes toxic.

The SOS (Salt Overly Sensitive) sensor complex is one of the most studied pathways for salt stress perception in Arabidopsis. The key components of this pathway are:

• SOS3 (CBL4)—a calcium-binding protein activated by an increase in cytosolic Ca²⁺ (which itself may be triggered by Na⁺ influx).
• SOS2 (CIPK24)—a protein kinase that binds to SOS3 and is activated by it.
• SOS1—a plasma membrane Na⁺/H⁺ antiporter that is phosphorylated by SOS2 and activated, pumping Na⁺ out of the cell (Taiz et al., 2023; Lambers and Oliveira, 2019).

Thus, the entry of Na⁺ (or, more precisely, its effect on membrane potential and calcium homeostasis) leads to the activation of a cascade that enhances Na⁺ efflux. This is an example of how the cell senses ionic imbalance and responds to it.

4.4. Heavy Metal Ions: A Special Case

Heavy metal ions (Cd²⁺, Pb²⁺, Cu²⁺, etc.) also represent a change in environmental composition, but their perception may differ from that of macronutrients. Heavy metals often mimic essential ions, using their transporters to enter the cell. For example, Cd²⁺ can enter through Zn²⁺ or Ca²⁺ transporters (Taiz et al., 2023).

Once inside the cell, heavy metal ions can directly interact with proteins, replacing essential ions (e.g., Fe²⁺ or Zn²⁺) in enzyme active sites, leading to their inactivation. This can also serve as a signal: enzyme inactivation can lead to the accumulation of intermediates, changes in redox state, or activation of stress genes. Additionally, heavy metals are potent catalysts of Fenton reactions, generating reactive oxygen species (ROS), which themselves act as signals (Paulsen, 1994; Taiz et al., 2023). Thus, changes in ionic composition can induce oxidative stress, which we discussed as an early signal (see Part 1).

4.5. Signal Integration: From Ions to Physiological Response

So, how does the cell "notice" a change in environmental composition?

1. Osmotic component: Changes in salt concentration lower water potential, causing water loss and decreased turgor—a primary signal perceived via mechanosensitive channels and changes in membrane tension (as in water deficit).

2. Ionic component: The ions themselves (especially Na⁺) can affect membrane potential, open ion channels, activate signaling pathways (e.g., the SOS pathway), and serve as substrates or regulators for transporters.

3. Secondary signals: Changes in ion fluxes, especially Ca²⁺, trigger calcium signaling, which activates protein kinases, phosphatases, NADPH oxidases, generating ROS and other secondary messengers.

4. Indirect effects: Heavy metal ions can directly damage proteins and generate ROS, which also act as signals.

This complex mechanism allows the cell not only to register the fact of a change in chemical composition but also to distinguish between different types of ions and their concentrations, tailoring the response accordingly.

In the next part, we will examine how the plant senses oxygen deficiency and why this signal arises before visible damage appears.

5.1. Oxygen as the Final Electron Acceptor in Respiration

First, let's recall that oxygen is required for the function of the mitochondrial electron transport chain (ETC). During oxidative phosphorylation, reduced carriers (NADH and FADH₂) donate electrons to the ETC, and the final acceptor of these electrons is molecular oxygen (O₂), which is reduced to water (Медведев, 2012; Lambers and Oliveira, 2019). This process—aerobic respiration—is the main source of ATP in plant cells, providing up to 95% of the energy consumed by the cell.

As long as oxygen is sufficient, electrons flow through the ETC, and the oxidized forms NAD⁺ and FAD are regenerated, maintaining the balance of redox pairs. When oxygen availability decreases, the ETC cannot transfer electrons further, and they begin to accumulate on previous carriers. This leads to the reduction of the carrier pool: the ratios NADH/NAD⁺ and FADH₂/FAD increase sharply (Медведев, 2012; Schopfer and Brennicke, 2016). This change in ratios is the change in the cell's redox state.

5.2. Redox State as the Primary Signal for Hypoxia

The redox state is not just an abstract indicator. It determines the activity of numerous enzymes, regulatory proteins, and transcription factors that are sensitive to the ratio of reduced and oxidized forms of coenzymes (e.g., NADH/NAD⁺). During hypoxia, the change in this ratio occurs before toxic metabolites (ethanol, lactic acid, acetaldehyde) accumulate or membranes are damaged. This is the primary signal for oxygen deficiency (Taiz et al., 2023).

The redox state can affect the cell in at least two ways:

1. Changing enzyme activity. Many enzymes of the Krebs cycle, glycolysis, and other metabolic pathways are regulated by the NADH/NAD⁺ ratio. During hypoxia, the accumulation of NADH inhibits citrate synthase, isocitrate dehydrogenase, and other enzymes, slowing down the Krebs cycle. This, in turn, stimulates glycolysis and a switch to enzymatic pathways that do not require oxygen (Медведев, 2012). Thus, the change in redox state immediately restructures metabolism.

2. Influence on regulatory proteins. Some sensor proteins, for example, transcription factors of the ERF (Ethylene Response Factor) family in plants, contain oxidation-sensitive amino acid residues that stabilize or destabilize the protein depending on oxygen concentration. In Arabidopsis and rice, group VII ERF proteins are degraded in the presence of oxygen via the N-end rule pathway, but are stabilized under hypoxia, activating the expression of genes encoding fermentation enzymes (Taiz et al., 2023). This is an example of direct oxygen level perception, albeit at the molecular level (see caveat in the condition). Nevertheless, the redox shift itself is a more fundamental and earlier signal.

5.3. Why Does the Redox Change Precede Damage?

Damage during prolonged hypoxia arises from several causes:

• Accumulation of toxic fermentation products (ethanol, acetaldehyde, lactic acid), which damage membranes and inhibit enzymes.
• Cytoplasmic acidification due to the formation of organic acids, disrupting pH-dependent enzymes.
• ATP deficiency → cessation of protein synthesis, transport disruption, loss of ion gradients.
• Production of reactive oxygen species (ROS) during reoxygenation after hypoxia, as well as within the ETC during partial oxygen reduction.

However, all these processes are secondary. They develop after the redox state has already changed. For instance, the switch to fermentation (alcoholic or lactic acid fermentation) is an adaptive response aimed at regenerating NAD⁺ from NADH, allowing glycolysis to continue producing at least 2 ATP per glucose molecule (Медведев, 2012). This response is triggered precisely by the change in redox state, not by the accumulation of damage. Thus, the change in the ratio of reduced/oxidized carriers is an "early warning" signal that activates protective mechanisms before the cell suffers from toxins or energy starvation.

5.4. Perception of Hypoxia via Metabolic Shifts

Interestingly, oxygen deficiency also indirectly affects other physiological processes. For example, during hypoxia, mitochondrial oxidative phosphorylation is inhibited, leading to the accumulation of ADP and inorganic phosphate. This stimulates glycolysis via the Pasteur effect (acceleration of glycolysis under ATP limitation) (Медведев, 2012). Furthermore, hypoxia activates ethylene synthesis, which in turn can induce the formation of aerenchyma and other adaptations to flooding (Schopfer and Brennicke, 2016). But again, these changes occur after the redox state has already shifted. Therefore, perception of hypoxia begins precisely with the disruption of electron flow in the respiratory chain.

5.5. Comparison with Other Types of Stress

It is important to note that in the case of oxygen deficiency, the primary signal is not a change in some physical structure (as for temperature or turgor), but a change in metabolic state—the ratio of reduced to oxidized coenzymes. This change occurs before any visible changes, such as mitochondrial swelling, membrane degradation, or the appearance of necrosis. Thus, the cell learns in advance about oxygen deficiency through changes in electron flows, even if the damage itself is not yet present. This is the answer to the question: because sensitive regulatory systems respond to changes in the NADH/NAD⁺ ratio and other redox pairs, which are a direct consequence of oxygen deficiency, rather than to the later consequences of this deficiency.

6.1. Reactive Oxygen Species: Inevitable Companions of Life in an Aerobic Environment

Let's start with a fundamental fact: in any cell that uses oxygen for respiration or photosynthesis, reactive oxygen species (ROS) are constantly formed—superoxide anion (O₂⁻•), hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), and singlet oxygen (¹O₂) (Sharma et al., 2012; Медведев, 2012). This is inevitable because in electron transport chains (mitochondrial, chloroplastic, microsomal), some electrons "leak" directly to oxygen, rather than completing full four-electron reduction to water. Normally, about 1–5% of all consumed oxygen is converted to ROS (Медведев, 2012). This means that ROS are not exotic but a constant background of cellular metabolism.

Plant cells possess a powerful antioxidant system (superoxide dismutases, catalases, peroxidases, ascorbate, glutathione, carotenoids, etc.) that normally effectively removes these ROS, maintaining their concentration at a low, non-toxic level (Sharma et al., 2012; Taiz et al., 2023). This balance between ROS production and scavenging maintains the cell's redox homeostasis.

6.2. Disruption of the Balance Under Stress: Conditions for the Oxidative Burst

When a plant encounters any stress—drought, salinity, extreme temperature, excess light, oxygen deficiency, or pathogen attack—the balance between ROS production and scavenging is disrupted. Why?

• Under water deficit, stomata close, CO₂ uptake decreases, and the photosynthetic electron transport chain becomes overloaded with electrons that cannot be used for CO₂ reduction. This leads to increased "leakage" of electrons to oxygen in the chloroplasts (Paulsen, 1994; Taiz et al., 2023).
• Under temperature stress (especially high temperature), enzyme activity and membrane fluidity change, which can disrupt the function of photosystems and mitochondrial complexes, promoting ROS formation (Paulsen, 1994; Lambers and Oliveira, 2019).
• Under salinity, excess Na⁺ can damage electron transport chains and inhibit antioxidant enzymes (Taiz et al., 2023).
• Under oxygen deficiency, electrons stall at intermediate carriers in mitochondria, and some of them reduce oxygen to superoxide; moreover, during reoxygenation after hypoxia, massive ROS production occurs (Медведев, 2012; Taiz et al., 2023).
• During pathogen attack, the plasma membrane NADPH oxidase is activated, specifically generating superoxide in the apoplast as part of the defense response (the so-called "respiratory burst") (Taiz et al., 2023).

Thus, virtually any stress increases ROS production and/or decreases antioxidant system activity. Therefore, the concentration of ROS in the cell starts to rise. This happens very quickly, often within seconds or minutes of the onset of the stress.

6.3. Why is This Not Just Damage, But a Signal?

It is important to understand: the initial increase in ROS levels during stress is not a catastrophe, but an alarm signal. Why?

Firstly, the ROS concentration initially rises not to a toxic level, but to a level that activates sensitive sensors. For instance, H₂O₂ at low (micromolar) concentrations can act as a signal, while toxic effects (membrane damage, protein oxidation) develop at much higher concentrations (Taiz et al., 2023; Sharma et al., 2012). Thus, the cell has a "margin of safety"—it can use ROS as a signal without dying.

Secondly, ROS can selectively oxidize specific proteins, altering their activity. For example, oxidation of thiol groups (–SH) in cysteine residues of certain transcription factors, protein kinases, and phosphatases can activate or inhibit them, causing changes in gene expression (Taiz et al., 2023). This is not random damage but a regulatory mechanism.

Thirdly, ROS can serve as signals for systemic information transmission throughout the plant. It is known that H₂O₂ and other ROS can spread through the apoplast, eliciting responses in remote organs. This is the basis of systemic acquired acclimation (SAA) during stress (Taiz et al., 2023).

Thus, the oxidative burst is not merely a consequence of damage, but an evolutionarily developed mechanism for rapid cell alerting to stress. It appears first because:

1. ROS are always-present metabolites that are continuously produced and removed.

2. Any stress immediately disrupts the balance of ROS production and removal.

3. The cell is sensitive to changes in ROS concentration through specialized sensor proteins and through changes in redox state.

4. ROS can rapidly activate signaling cascades (calcium, phosphorylation) and alter gene expression.

6.4. Mechanisms of the Oxidative Burst: Sources of ROS

Where do ROS come from during stress? The main sources are:

• Chloroplasts. Under excess light or CO₂ limitation, electrons are transferred from photosystem I to oxygen (Mehler reaction), forming superoxide. Singlet oxygen can also be formed during damage to photosystem II (Paulsen, 1994; Lambers and Oliveira, 2019).
• Mitochondria. In the respiratory chain, some electrons can reduce oxygen to superoxide at complexes I and III. Especially high amounts of ROS are produced when the ETC is damaged or during reoxygenation after hypoxia (Медведев, 2012).
• Apoplast and plasma membrane. NADPH oxidases (RBOH proteins) specifically generate superoxide in response to many stresses, especially during pathogen attack and osmotic stress. This is an active, regulated process (Taiz et al., 2023; Lambers and Oliveira, 2019).
• Peroxisomes and glyoxysomes. Oxidative reactions occur in these organelles (photorespiration, β-oxidation of fatty acids) that produce H₂O₂ (Медведев, 2012).
• Cell wall. Cell wall peroxidases can generate ROS in response to mechanical damage or pathogens (Taiz et al., 2023).

6.5. Why Does the Oxidative Burst Appear Before Other Responses?

Let's compare the speed of the oxidative burst with other signals:

• Change in turgor—a physical process, can occur in seconds, but is specific to water stress.
• Change in membrane fluidity—a rapid physical response, but also specific to temperature.
• Redox shift due to oxygen deficiency—rapid, but specific to hypoxia.
• Oxidative burst—a universal response that occurs in virtually any stress. Because any stress disrupts electron transport or antioxidant defense in one way or another. And since ROS are already present in the cell, their concentration begins to rise immediately upon the emergence of an imbalance.

Furthermore, the oxidative burst can occur deliberately, as an actively regulated process (via NADPH oxidases). It is not just a passive consequence of stress but part of a genetically programmed response.

6.6. The Oxidative Burst as a Signal Integrator

Interestingly, ROS can interact with other secondary signals. For example, H₂O₂ can activate calcium channels, leading to an increase in cytosolic Ca²⁺. In turn, increased Ca²⁺ can activate NADPH oxidase, generating even more ROS. This creates a positive feedback loop that rapidly amplifies the signal (Taiz et al., 2023). This allows even a weak stress to trigger a strong signaling response.

Thus, the oxidative burst is the first universal distress signal because:

• ROS are constant companions of aerobic metabolism.
• Any stress immediately disrupts the balance between ROS production and removal.
• The cell can sense this change via specialized sensors.
• ROS can rapidly activate multiple protective pathways.
• The oxidative burst can be an actively regulated process (via NADPH oxidases), not just a passive consequence.

Final Summary of the Lecture.

We have examined five main types of primary signals that allow a plant to detect changes in environmental conditions:

1. Water deficit → decreased turgor → change in mechanical tension of membrane and cell wall → activation of mechanosensitive channels.

2. Temperature stress → change in lipid bilayer fluidity → change in activity of membrane proteins → generation of biochemical signals.

3. Change in ionic composition → change in membrane potential, direct interaction of ions with proteins, activation of transporters → triggering signaling cascades (calcium, SOS pathway).

4. Oxygen deficiency → shift in redox state (NADH/NAD⁺, FADH₂/FAD) → metabolic reprogramming and activation of adaptive programs.

5. Oxidative burst → increased ROS concentration (superoxide, hydrogen peroxide) → universal alarm signal, interacting with other signaling systems.

All these primary signals arise before visible damage develops, serving as early warnings for the plant, allowing it to launch protective mechanisms preemptively.

In the following lectures, we will explore how these primary signals are converted into secondary messengers (calcium, ROS, protein phosphorylation) and how they trigger specific response programs—from osmoprotectant synthesis to morphological changes and stomatal closure.