Signal pathways and system communication

1.1. A language understood by every cell

When we talk about signaling systems, we are essentially talking about the language in which cells communicate with each other and with the external environment. And if in animals such a universal language is often nerve impulses, in plants this role is played by secondary messengers—small molecules or ions whose concentration inside the cell changes rapidly in response to an external stimulus.

The most studied and perhaps the most universal secondary messenger in plants is calcium ions (Ca²⁺). Why calcium? The answer lies in its unique properties and in how the plant cell is organized.

At rest, in the absence of any stress influences, the concentration of free calcium ions in the cytosol is extremely low—on the order of 50–100 nanomoles (Taiz et al., 2023). This is many times less than the calcium concentration in the cell wall, the vacuole, or the endoplasmic reticulum. In other words, the cell maintains a huge concentration gradient of calcium between the cytosol and various storage compartments.

This situation resembles a coiled spring. As soon as calcium channels in the membranes open, calcium literally rushes into the cytosol, raising its concentration tenfold or even hundredfold within seconds or minutes (Taiz et al., 2023; Lambers & Oliveira, 2019). Such a rapid and powerful surge is an ideal alarm signal. It does not require the synthesis of new molecules, it is almost instantaneous, and it directly tells the cell: "something unusual has happened."

1.2. How does the cell read the calcium signal?

But the calcium concentration surge itself is only the first part of the story. The second, no less important part is how the cell interprets this surge. Here, sensor proteins capable of binding calcium ions and changing their structure and activity come onto the scene.

The chief among these sensors is calmodulin—a small, incredibly conserved protein found in almost all eukaryotes (Lambers & Oliveira, 2019). When calcium binds to calmodulin, the protein changes its conformation, "turns on," and gains the ability to interact with many other target proteins—primarily protein kinases.

In addition to calmodulin, plants possess an entire family of calcium-dependent protein kinases (CDPKs) —enzymes unique to plants and some protists that combine in a single molecule the function of calcium binding and protein phosphorylation (Taiz et al., 2023; Lambers & Oliveira, 2019). This makes them ideal "translators" of the calcium language into the language of phosphorylation—one of the most common ways to regulate protein activity in the cell.

Another group of sensors is calcineurin B-like proteins (CBLs) , which, upon binding calcium, interact with their partners—CBL-interacting protein kinases (CIPKs) —forming signaling modules responsible for specific responses to various stresses (Taiz et al., 2023).

1.3. The calcium signal is not just "on/off"

It is important to emphasize a key idea here: the calcium signal is not a binary code with only "signal present" and "no signal." The cell is able to distinguish the amplitude, frequency, and spatial localization of calcium spikes. This phenomenon is called calcium coding (Taiz et al., 2023).

Imagine that different stresses—drought, salinity, cold, mechanical damage—cause different patterns of calcium concentration fluctuations: somewhere it will be a rapid single peak, somewhere a series of damped oscillations, somewhere a prolonged plateau. And each of these "patterns" can be recognized by a specific set of sensors and trigger a unique response program (Lambers & Oliveira, 2019).

This principle—specificity through universality—underlies all of plant signaling physiology. One and the same signaling language, but different "dialects," understood by different executive systems.

1.4. Example: calcium and stomatal closure

To make this abstraction more tangible, let us consider a classic example—stomatal closure under the action of abscisic acid (ABA) during water deficit (Taiz et al., 2023).

When roots sense that the soil is drying out, they synthesize ABA. This hormone, carried by the water flow, reaches the leaves and arrives at the stomatal guard cells. There, the following occurs:

1. ABA binds to its receptor on the plasma membrane.

2. This triggers the opening of calcium channels, and the Ca²⁺ concentration in the cytosol of guard cells rises sharply—from about 50–100 nM to 500–1100 nM (Taiz et al., 2023).

3. The rise in calcium leads to the opening of anion channels—Cl⁻ and malate ions leave the cell, causing membrane depolarization.

4. Depolarization, in turn, opens potassium channels, and potassium also exits the cell.

5. The efflux of ions leads to water loss, guard cell turgor drops, and stomata close.

Notice: throughout this entire chain, calcium acts as a switch that triggers a whole cascade of events. It is the first alarm signal telling the cell: "start closing."

1.5. Why is calcium universal rather than specific?

If one and the same ion participates in responses to dozens of different stresses, a legitimate question arises: how does the cell not confuse a drought signal with a pathogen attack signal?

The answer lies in the fact that calcium is a tool, not the content of the message. Its role is to quickly convey the information that something has changed. The specific content—what kind of stress, how to respond to it—is determined by the "context":

• Other signals arriving simultaneously with the calcium signal—for example, reactive oxygen species or hormones.
• The sensor apparatus of the particular cell—which receptors and channels it possesses.
• The program already "written" in the cell's genome and determined by its differentiation and physiological state.

Calcium is a universal language in which the cell talks about stress. But the meaning of the phrase depends on who, when, and in what context speaks it (Lambers & Oliveira, 2019).

1.6. From cell to organism

Remarkably, the calcium signal is not confined within a single cell. It has been shown that under local stress, for example, when one leaf is exposed to excessively bright light, a wave of elevated calcium concentration propagates throughout the plant at a rate of approximately 1–4 cm per minute (Taiz et al., 2023). This phenomenon is called a calcium wave. It moves from cell to cell through plasmodesmata and possibly with the involvement of the apoplast, "warning" all organs that an adverse event has occurred somewhere.

The calcium wave is the first level of systemic communication, which we will discuss in more detail toward the end of the lecture. But it is already important to understand now: calcium is not just an intracellular signal. It is a means of rapid information transfer between cells and tissues.

Section summary

So, colleagues, we have seen that calcium ions are a universal language of stress signaling in plants. Their key properties are:

1. Rapidity—the concentration change occurs within seconds.

2. Power—the concentration gradient allows for a strong signal.

3. Recognizability—calcium binds to special sensor proteins that translate its signal into changes in the activity of other proteins.

4. Informativeness—the nature of the calcium signal (amplitude, frequency, localization) carries information about the nature of the stress.

5. Systemicity—calcium waves can propagate throughout the organism.

Calcium is the first line of defense, the fastest way to tell the cell: "Attention, something is happening!" But this signal alone does not yet determine the response. The response is formed at the following levels, where other players join calcium. And we will discuss one of them—reactive oxygen species—in the next part of our lecture.

2.1. Inevitable companions of life in an oxygen atmosphere

Colleagues, we have established that calcium is a universal alarm signal. But the calcium spike itself is not yet the full story nor a treatment plan. It is merely a "call" requiring further interpretation. And here other players come onto the scene, whose role is both critical and paradoxical. We are talking about reactive oxygen species (ROS).

Let us reflect. Plants are aerobic organisms. They live in an atmosphere containing about 21% oxygen. They themselves produce oxygen during photosynthesis. And this very oxygen, without which life is impossible, under certain conditions becomes a source of serious danger.

Molecular oxygen (O₂) in its ground state is relatively inert. But during cellular metabolism, it can be reduced not completely, but stepwise, capturing one electron at a time. As a result, highly reactive intermediates are formed: superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), as well as singlet oxygen (¹O₂) —an excited form of O₂ formed by energy transfer from excited chlorophyll molecules (Schopfer & Brennicke, 2016; Pessarakli, 2020).

These molecules and radicals possess high chemical reactivity. In physiology, they are collectively termed "reactive oxygen species" (ROS). And here we encounter a fundamental contradiction: ROS are both necessary and deadly dangerous. The plant's task is not to eliminate them entirely, but to maintain their level within strictly defined physiological limits.

2.2. The "dark side" of ROS: oxidative stress and damage

When a plant finds itself under stressful conditions—whether drought, salinity, extreme temperatures, or pathogen attack—the balance in the cell is disrupted. Electron transport chains in chloroplasts and mitochondria become overloaded, and the electron flow begins to "leak" to oxygen to a greater extent, generating ROS (Lambers & Oliveira, 2019; Taiz et al., 2023).

This process is not merely a side effect. Under intense stress, the rate of ROS formation increases many times over. And if the cell's defense systems cannot cope, a state called oxidative stress ensues.

What makes oxidative stress dangerous? ROS are powerful oxidizers. They attack all major classes of biomolecules:

• Lipids—peroxidation of polyunsaturated fatty acids in membranes is triggered. This disrupts membrane fluidity and barrier properties, leads to ion and metabolite leakage, and disorganizes the function of membrane proteins (Pessarakli, 2020; Schopfer & Brennicke, 2016).
• Proteins—oxidation of amino acid residues (especially cysteine, methionine, histidine, tryptophan) leads to loss of enzymatic activity, formation of carbonyl groups, aggregation, and protein degradation.
• Nucleic acids—ROS cause DNA strand breaks, base modifications (e.g., formation of 8-hydroxyguanine), which can lead to mutations and transcriptional disruption (Schopfer & Brennicke, 2016).

Especially dangerous is the hydroxyl radical (•OH) —it is so reactive that the cell has no enzymatic mechanisms for its neutralization. The only strategy is to prevent its formation by preventing the accumulation of H₂O₂ in the presence of transition metal ions (the Fenton reaction) (Lambers & Oliveira, 2019).

When damage becomes irreversible, the cell may trigger programmed cell death (apoptosis)—a kind of "sacrifice" for the sake of saving the whole organism (Medvedev, 2012).

2.3. The "bright side": ROS as signaling molecules

But plant physiology would be too simplistic if ROS carried only destruction. Evolution found an amazing solution: moderate levels of ROS are a signal, not a sentence.

A key role here is played by hydrogen peroxide (H₂O₂) . Unlike the short-lived superoxide and hydroxyl radical, H₂O₂ is relatively stable, can diffuse through membranes, and travel significant distances within and between cells (Taiz et al., 2023). This makes it an ideal signaling messenger.

How can H₂O₂ serve as a signal? First, it can directly oxidize thiol groups (-SH) of cysteines in regulatory proteins, altering their conformation and activity. This mechanism underlies the function of many transcription factors and protein kinases sensitive to the redox status of the cell (Lambers & Oliveira, 2019).

Second, H₂O₂ activates calcium channels, causing additional Ca²⁺ influx into the cytosol. Conversely, calcium activates NADPH oxidases—enzymes that generate superoxide anion, the precursor of H₂O₂. This creates a positive feedback loop: calcium → activation of NADPH oxidase → ROS production → opening of calcium channels → even more calcium (Taiz et al., 2023). This mechanism allows the signal to be rapidly amplified and spread throughout the cell, and then throughout the plant.

Moreover, H₂O₂ acts as one of the central regulators of defense gene expression. It activates transcription factors that "switch on" the synthesis of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase), chaperones (heat shock proteins), as well as enzymes for the biosynthesis of protective compounds (phenolic compounds, phytoalexins) (Schopfer & Brennicke, 2016; Medvedev, 2012).

2.4. The concept of the "oxidative window": physiology as a search for optimum

Here we come to a key idea that distinguishes the modern understanding of stress physiology from the old, simplistic view of "ROS = evil." This idea is called the concept of the oxidative window (or redox buffer).

Its essence is that the plant does not attempt to eliminate all ROS to zero. This would be impossible and meaningless. Instead, the cell maintains the ROS concentration within a certain optimal range, called the "window":

• Below the window—the signal is too weak, defense systems are not activated, the plant remains vulnerable to stress.
• Within the window—ROS perform a signaling function, triggering adaptive programs that enhance resistance.
• Above the window—oxidative stress ensues, damage becomes irreversible, and the cell dies.

This balance is maintained by a powerful antioxidant system that operates as a single conveyor (Kuznetsov, 2006; Pessarakli, 2020). It includes:

• Low-molecular-weight antioxidants: ascorbate (vitamin C), glutathione, α-tocopherol (vitamin E), carotenoids, flavonoids.
• Antioxidant enzymes: superoxide dismutase (SOD)—converts superoxide to H₂O₂; catalase (CAT) and ascorbate peroxidase (APX)—decompose H₂O₂ to water; as well as enzymes of the so-called ascorbate-glutathione cycle (dehydroascorbate reductase, glutathione reductase), which regenerate the reduced forms of antioxidants (Taiz et al., 2023; Schopfer & Brennicke, 2016).

It is important to understand that this system does not simply "remove" ROS. It keeps them at a signaling level. When stress intensifies, ROS production increases, and the antioxidant system is activated to prevent crossing the upper boundary of the window. If stress weakens, antioxidant activity decreases, and the ROS level returns to basal, but does not drop to zero.

2.5. ROS and systemic communication: the oxidative signal wave

And finally, another surprising fact: like calcium waves, ROS can spread systemically throughout the plant. During local stress (for example, when one leaf is illuminated or damaged), a burst of H₂O₂ is generated at the site of exposure. This oxidative signal wave then propagates through the plant at a rate of about 1–2 cm per minute, affecting distant tissues (Taiz et al., 2023).

This process is called a systemic ROS wave (or ROS wave). It moves from cell to cell, probably through plasmodesmata and the apoplast, and its propagation requires the involvement of NADPH oxidases (RBOH proteins). Each successive cell along the wave activates its own NADPH oxidases, generating ROS and passing the signal further (Lambers & Oliveira, 2019).

Thus, ROS perform not only intracellular signaling roles but also act as intercellular and even systemic messengers. They interact with calcium waves, reinforcing and sustaining each other, forming a unified signaling network that rapidly "alerts" the entire organism to local stress.

2.6. Summary: from destroyers to signal builders

Colleagues, let us summarize. Reactive oxygen species are a classic example of the dual nature of physiological signals. Depending on concentration and context, the same molecules can act both as deadly weapons and as essential tools for adaptation.

Key conclusions:

1. ROS are inevitable products of metabolism, especially under stress.

2. At high concentrations, they cause oxidative stress: damage lipids, proteins, DNA, leading to cell death.

3. At low (signaling) concentrations, they activate defense genes, promote stomatal closure, and participate in systemic signal transmission.

4. The plant maintains ROS levels within the "optimal window" using a powerful antioxidant system that does not eliminate ROS completely but regulates their concentration.

5. ROS closely interact with calcium signals, forming positive feedback loops that amplify and spread the signal throughout the organism.

Thus, stress physiology is the science of maintaining balance between destruction and signal, between stress and adaptation. And ROS are one of the most striking examples of how a plant uses potentially dangerous molecules for its own survival.

In the next part, we will look at how the cell amplifies and transforms these signals through protein kinase cascades to translate the alarm into the language of specific physiological changes.

3.1. The problem of the weak signal

Colleagues, we already know: when a plant encounters stress, rapid changes occur in the cell—a calcium spike, a burst of reactive oxygen species. But the question is: is this enough to trigger a full-fledged adaptation program? Imagine you hear a faint sound—you need not just to hear it, but to understand what kind of sound it is, where it is coming from, and what to do in response. For this, the signal needs to be amplified, recognized, and converted into instructions understandable to the cell.

And here we come to one of the most elegant systems of cellular signaling—protein kinase cascades, which act as biological signal amplifiers.

3.2. Protein kinases—a universal regulatory tool

What are protein kinases? They are enzymes that transfer a phosphate group from ATP to specific amino acid residues in target proteins. Most often, phosphorylation occurs on serine, threonine, and tyrosine.

Why is this needed? Phosphorylation causes conformational changes in the protein. Imagine a protein as a complex molecular machine with many moving parts. Attaching a phosphate group is like pulling a lever or turning an ignition key. The protein changes its spatial structure, and this can:

• activate or inactivate its enzymatic activity;
• change its ability to bind to other molecules (substrates, cofactors, regulatory proteins);
• affect its intracellular localization (for example, "move" the protein from the cytosol to the nucleus or vice versa);
• change its lifetime in the cell.

The most important property of this mechanism is reversibility. There are enzymes that phosphorylate proteins (protein kinases), and enzymes that remove the phosphate group—protein phosphatases. The cell uses this as a constantly operating "toggle switch," finely regulating the activity of thousands of proteins in response to ever-changing conditions (Taiz et al., 2023; Lambers & Oliveira, 2019).

3.3. The MAPK cascade: a relay race of signal transmission

Among the multitude of protein kinases, a special place is occupied by the MAPK (Mitogen-Activated Protein Kinases) family. This name is historical, but in plants, MAPKs participate not only in the regulation of cell division but also in responses to almost all known stresses: drought, salinity, high and low temperatures, pathogen attack, mechanical damage (Taiz et al., 2023).

What makes the MAPK cascade so effective? It is its multi-step structure. This is not a single reaction, but a sequence of events working on the principle of a relay baton:

1. The first step—MAP3K (or MAPKKK). This is the first kinase in the cascade. It is activated in response to the arrival of the primary signal—a calcium spike, elevated H₂O₂ levels, hormone binding to a receptor. MAP3K phosphorylates and activates the next kinase.

2. The second step—MAP2K (or MKK). This kinase specifically phosphorylates MAPK, necessarily at two amino acid residues—threonine and tyrosine, separated by one amino acid (the so-called TXY motif). This double phosphorylation is a prerequisite for full activation of MAPK.

3. The third step—MAPK. This is the terminal kinase in the cascade. It phosphorylates many target proteins, among which are transcription factors, other kinases, enzymes, cytoskeletal proteins. It is MAPK that is the "executive mechanism" that directly changes the physiological state of the cell.

Imagine that each activated kinase can phosphorylate not one but several molecules of the next kinase. This creates a cascade amplification effect: one signal at the input turns into thousands of modified proteins at the output (Taiz et al., 2023; Pessarakli, 2020).

3.4. From signal to program: how the cascade creates specificity

It would seem that if one cascade amplifies one signal and another amplifies another, how does the cell distinguish which specific signal arrived? How not to confuse a drought signal with a pathogen signal?

Several levels of specificity operate here.

The first level—receptor-based. Different stresses activate different MAP3Ks because they are coupled to different receptors and sensors. For example, cold activates one set of MAPK pathways, while osmotic stress activates others.

The second level—modular. Different cell types and different physiological states express different sets of MAPKs. The same signal can elicit different responses in root and leaf.

The third level—spatial. MAPKs can be localized in different compartments of the cell: some cascades operate in the cytosol, some in the nucleus, some are membrane-associated. This creates an additional dimension of specificity (Lambers & Oliveira, 2019).

And finally, the fourth level—temporal. The speed and duration of MAPK cascade activation also carry information. A fast, short-lived signal may trigger one program, while a prolonged, gradually decaying one may trigger a completely different one. This resembles calcium coding, but at the level of phosphorylation (Schopfer & Brennicke, 2016).

3.5. MAPK and ROS: a signal that amplifies a signal

Particularly interesting is the interaction of MAPK cascades with reactive oxygen species. We have already discussed that H₂O₂ can act as a signaling molecule. It turns out that MAPK cascades can be activated both by H₂O₂ and, conversely, can trigger its production.

How does this work? One group of MAPK targets is NADPH oxidases (RBOH proteins) , which generate superoxide anion, the precursor of H₂O₂. Phosphorylation of these enzymes by MAPK cascades enhances their activity, leading to an additional burst of ROS (Taiz et al., 2023; Lambers & Oliveira, 2019).

This creates a positive feedback mechanism: stress → calcium → MAPK activation → NADPH oxidase phosphorylation → enhanced ROS production → ROS activates new MAPK cascades → signal escalates.

This loop is one of the key mechanisms for amplifying and spreading the stress signal. It allows a local event to turn into a powerful signal that can be transmitted over long distances.

3.6. Why it is important to talk about amplification rather than just transmission

Colleagues, it is important to emphasize the fundamental difference here. The plant's signaling system is not simply a "wire" or a "telephone line" through which information is transmitted unchanged. It is an active, energy-dependent system of amplification and transformation.

Each stage of the cascade can:

• amplify the signal—one molecule of activated kinase can phosphorylate many molecules of the next;
• modulate the signal—at each stage, regulation is possible, a "tuning" of the response intensity;
• integrate signals—at the level of MAP2K or MAPK, signals from different primary sensors can converge, creating a complex, multidimensional response;
• create memory—some phosphorylations can persist for a long time, providing "memory" of the stress experienced.

3.7. From amplification to action: MAPK and transcription factors

And finally, the most important step—activation of transcription factors. MAPK cascades phosphorylate specific transcription factors, which then move into the nucleus, bind to regulatory regions of DNA, and trigger the transcription of hundreds of defense genes (Medvedev, 2012; Kuznetsov, 2006).

Among these genes:

• genes for antioxidant enzymes (SOD, CAT, APX)—to keep ROS within the "window";
• genes for osmolyte synthesis enzymes (proline, glycine betaine, sugars)—for osmotic adaptation;
• genes for heat shock proteins (HSP)—to protect against protein denaturation;
• genes for ABA synthesis enzymes—to trigger hormonal signaling;
• genes for PR proteins and phytoalexins—for protection against pathogens.

Thus, MAPK cascades act as a bridge between primary signal perception and the reprogramming of the genetic program, ensuring a rapid and adequate response to stress.

3.8. Summary: amplification as the basis of adaptation

So, colleagues, we have seen that signal amplification is not just a technical detail. It is the basis of adaptation. Without cascade amplification, the cell could not transition from rapid but weak signals (Ca²⁺ spike, ROS burst) to slow but powerful programs of gene expression change, growth, and development.

Key conclusions:

1. Protein kinases are universal regulators that transmit signals through phosphorylation, altering the activity, localization, and interactions of target proteins.

2. The MAPK cascade is a three-tier system (MAP3K → MAP2K → MAPK) that works like a relay race and provides multiple signal amplification.

3. Specificity is achieved through a combination of receptors, cell types, spatial localization, and temporal characteristics of activation.

4. Interaction with ROS creates positive feedback loops, amplifying and spreading the signal throughout the cell and organism.

5. The ultimate targets of MAPKs are transcription factors that switch the cell's genetic program, triggering the synthesis of protective proteins.

Now that we know how signals are amplified and transformed inside the cell, we are ready to move to the next level of integration—hormonal regulation, where individual signaling pathways converge into a coordinated response of the whole organism.

4.1. From intracellular signals to intercellular communication

Colleagues, we have already come a long way. We saw how a cell perceives stress through a calcium spike, how reactive oxygen species serve simultaneously as both signal and threat, and how protein kinase cascades amplify this signal many times over, preparing the ground for changes in the genetic program.

But all of this happened inside a single cell. Now imagine the scale of the problem. A plant may have thousands of leaves, kilometers of roots, millions of cells. Stress may arise at one point—for example, a root has dried out, or a caterpillar has eaten a leaf. But the response must be systemic: stomata in other leaves close, root growth accelerates or slows, dormancy programs are launched in growth points. How is this complex orchestra coordinated?

The answer—hormones. It is hormones that become the signaling molecules capable of traveling over long distances and linking together the responses of different organs. And what is especially important, hormones distribute roles among different parts of the plant, creating an integrated survival strategy.

4.2. Hormones as conductors, not soloists

Here we come to the most important methodological principle: hormones do not act in isolation, but in complex interaction networks. They do not simply "turn on" or "turn off" certain processes. They create the context in which other signals acquire specific meanings.

A classic example is the antagonism of cytokinins and abscisic acid (ABA) (Taiz et al., 2023; Kuznetsov, 2006). Under normal water supply, cytokinins predominate in the plant, supporting growth, cell division, and delaying leaf senescence. As soon as roots sense soil drying, cytokinin synthesis drops and ABA production rises sharply. ABA begins to dominate—and the entire plant physiology is restructured: stomata close, shoot growth is inhibited, root growth is activated in search of water.

But it is not that cytokinins are "growth hormones" and ABA is a "stress hormone." This is too simplistic a picture. What matters is the ratio of hormones. The same hormone can cause different effects depending on which other hormones it interacts with and in what physiological context it acts (Kuznetsov, 2006).

4.3. ABA—an integrator of water deficit

Abscisic acid is perhaps the most studied stress hormone in plants. Its role in water deficit we have already partially discussed, but now let us look at it as a tool of integration (Lambers & Oliveira, 2019; Pessarakli, 2020).

When roots enter dry soil, they synthesize ABA. This hormone rises with the transpiration stream to the leaves, where it:

• Closes stomata—we have already examined this mechanism with calcium involvement.
• Switches the genetic program—ABA activates transcription factors that trigger the synthesis of hundreds of protective proteins, including LEA proteins, dehydrins, and osmolyte synthesis enzymes.
• Inhibits shoot growth—conserves resources for survival.
• Stimulates root growth—directs assimilates to the underground part, increasing the water-absorbing surface.

But ABA does not work alone. Its signal is modulated by other hormones. For example, cytokinins can attenuate the effect of ABA on stomata, while ethylene can enhance or attenuate it depending on concentration and physiological state. This creates fine-tuning of the response: under mild water deficit, the plant may maintain some growth; under severe deficit, it switches entirely to survival.

4.4. Ethylene, jasmonic acid, and salicylic acid—defense against damage and pathogens

Another group of hormones is associated with responses to mechanical damage and pathogen attack. Here, key roles are played by ethylene, jasmonic acid (JA), and salicylic acid (SA) (Medvedev, 2012; Schopfer & Brennicke, 2016).

The interaction scheme of these hormones resembles a crossroads where each signal chooses its path:

• Salicylic acid is mainly associated with responses to biotrophic pathogens (those that feed on living cells, e.g., viruses, some fungi, and bacteria). It participates in the formation of systemic acquired resistance (SAR) and triggers the synthesis of PR proteins (pathogenesis-related proteins), including chitinases and β-1,3-glucanases, which destroy pathogen cell walls (Medvedev, 2012).

• Jasmonic acid and ethylene mainly respond to necrotrophic pathogens (those that kill cells and feed on dead tissue) and insect herbivores. They activate the synthesis of protease inhibitors, lectins, and phytoalexins—low-molecular-weight compounds with antimicrobial activity (Medvedev, 2012).

But perhaps the most interesting is the cross-regulation between these pathways. It has been established that SA and JA often act antagonistically: activation of an SA-dependent response can suppress a JA-dependent one, and vice versa (Medvedev, 2012). This allows the plant to choose the most appropriate defense strategy depending on the nature of the enemy.

Imagine: a plant is attacked by a necrotrophic fungus. It "understands" that it needs to activate the JA pathway to kill the pathogen along with the damaged cells. If it simultaneously turned on the SA pathway with its PR proteins, defense effectiveness might decrease because resources would be dispersed. Therefore, the plant has mechanisms that allow it to make a decision and focus on the main task. This is the "distribution of roles" at the level of hormonal regulation.

4.5. Hormonal balance as the language of integration

So, the key idea of this section is hormones do not command, they coordinate. They create integral signals that reflect the overall physiological state of the plant. What matters is not the amount of an individual hormone, but the balance between hormones, which determines the nature of the response.

This approach is called hormonal balance. It assumes that the physiological state of the plant is described not as the result of one hormone's action, but as the result of competitive and synergistic interactions between them (Kuznetsov, 2006).

How does this work in practice?

• The ABA/cytokinin ratio determines water-use strategy: high ABA and low cytokinins—water conservation mode; low ABA and high cytokinins—growth.
• The JA/SA ratio determines pathogen defense strategy: JA dominance—defense against necrotrophs and insects; SA dominance—defense against biotrophs.
• The interaction of ABA, ethylene, and jasmonates determines the response to complex stresses, such as a combination of drought and salinity.

4.6. Hormones and systemic signaling: fast and slow pathways

It is important here to make another distinction. Hormonal regulation operates on two timescales.

The fast pathway—when the hormone is already synthesized and transported through the plant with the water flow or through the phloem. For example, during rapid soil drying, ABA travels from roots to leaves within minutes. This pathway provides an immediate response.

The slow pathway—when stress induces the de novo synthesis of the hormone in the organs that directly experience it. This hormone can then be transported systemically or trigger the synthesis of other hormones in distal parts of the plant. This pathway provides adaptation to prolonged stresses (Taiz et al., 2023).

Interestingly, hormones can interact with calcium and ROS signals, creating a unified integrated network. For example, ABA, as we have seen, triggers a calcium spike in guard cells and launches ROS production. In turn, ROS can influence the synthesis of ABA and other hormones. Thus, regulatory loops arise where fast signals (Ca²⁺, ROS) and slow signals (hormones) mutually reinforce and modulate each other (Lambers & Oliveira, 2019).

4.7. Example: interaction of ABA and ethylene under salinity

To make this abstract discussion more concrete, let us consider a complex example—the plant's response to salinity. This stress combines an osmotic component (like drought) and an ionic component (Na⁺ and Cl⁻ toxicity).

Under salinity, ABA levels rise in the plant, leading to stomatal closure. But this is only the first line of defense. In parallel, ethylene synthesis is activated, which can:

• stimulate the formation of aerenchyma—air spaces that facilitate root respiration;
• activate the synthesis of proteins involved in ion compartmentalization into the vacuole;
• modulate the expression of genes responsible for osmolyte synthesis.

The ABA/ethylene ratio determines whether the plant will mainly "conserve water" or simultaneously "adapt to ionic stress." In some species, the ABA response dominates; in others, the ethylene-dependent one. This is the "distribution of roles" at the level of taxonomic and ecological strategies (Kuznetsov, 2006).

4.8. Summary: hormones as signal integrators

Colleagues, let us summarize. Hormones in the stress signaling system perform several critical functions:

1. Long-distance communication—hormones can move through the plant, linking the responses of roots, leaves, stems, and growth points.

2. Signal integration—they combine information from different sensors (calcium, ROS, membrane potential) into unified regulatory programs.

3. Role distribution—different hormones and their ratios determine which specific response will be activated under given conditions.

4. Temporal coordination—they link fast (seconds to minutes) and slow (hours to days) components of the stress response.

5. Cross-regulation—antagonism and synergy between hormones create a flexible decision-making system.

Thus, hormones are the conductors of the physiological orchestra. They do not play the music themselves, but determine which instruments and at what moment should sound. And it is through the hormonal system that local signals are transformed into a coordinated response of the entire organism.

5.1. From a local event to a systemic response

Colleagues, we have traveled a long road. We began with how an individual cell perceives a signal through a calcium spike, then saw how reactive oxygen species serve simultaneously as both danger and signal, examined how protein kinase cascades amplify this signal, and finally understood how hormones integrate these signals at the whole-organism level.

But the main question we posed at the beginning of the lecture remains: how does a local injury transform into a response of the whole plant? Why, for example, when roots encounter dry soil, do stomata in leaves close? Why, when one leaf is attacked by a pathogen, do other leaves become more resistant?

The answer lies in the field of systemic physiology—the science of how individual parts of a plant interact with each other, forming a unified, integrated organism. And today we will see that all the previously discussed mechanisms—calcium, ROS, MAPK cascades, hormones—work in a single network, providing a systemic response.

5.2. Three pillars of systemic signaling

Modern research shows that the plant's systemic response to stress is provided by three interconnected mechanisms (Taiz et al., 2023; Lambers & Oliveira, 2019):

1. Systemic reactive oxygen species wave (ROS wave) —a fast signal propagating through the apoplast and via plasmodesmata.

2. Systemic calcium wave—a signal closely linked to the ROS wave and propagating in a similar manner.

3. Systemic hormonal signals—slower but longer-lasting signals transmitted through the phloem and xylem.

All three mechanisms work in concert, creating a unified system of rapid alert and long-term physiological reprogramming.

5.3. The ROS wave: a fast alarm signal

As we have already discussed, under local stress (for example, when one leaf is exposed to excessively bright light or mechanically damaged), a burst of reactive oxygen species is generated at the site of exposure (Lambers & Oliveira, 2019; Taiz et al., 2023).

But the most surprising thing is that this signal does not remain localized. It propagates from cell to cell at a rate of about 1–4 cm per minute. How does this happen?

A key role here is played by NADPH oxidases (RBOH proteins) . When the cell at the injury site generates ROS, this causes activation of RBOH proteins in neighboring cells. These, in turn, begin to produce their own ROS. This creates a self-sustaining wave that can propagate over significant distances—from one leaf to another, from root to shoot (Taiz et al., 2023).

This has been experimentally demonstrated using fluorescent dyes sensitive to ROS. When one leaf of Arabidopsis was subjected to local stress, within a few minutes the oxidative signal wave reached distal leaves. In mutants with disrupted RBOH genes, such a wave did not occur, and the systemic response did not develop (Taiz et al., 2023).

It is important to understand that the ROS wave is not just "information that something has happened." It is a signal that itself triggers protective programs in distal tissues, preparing them for possible stress.

5.4. The calcium wave: a partner of the ROS signal

The ROS wave does not exist in isolation. It is closely linked to the calcium wave—the propagation of elevated Ca²⁺ concentration from cell to cell.

We have already discussed the mechanism of this link: ROS activate calcium channels, causing Ca²⁺ influx into the cytosol. And calcium, in turn, activates RBOH proteins, enhancing ROS production. Thus, a positive feedback loop arises that sustains and amplifies the propagation of both signals (Taiz et al., 2023; Lambers & Oliveira, 2019).

Interestingly, the calcium wave can precede the ROS wave or follow it, depending on the type of stress and the plant's physiological state. But in any case, these two signals work as a single systemic signaling module.

5.5. Hydraulic and electrical signals: faster than hormones

In addition to ROS and calcium, there are other mechanisms of rapid systemic information transmission. In particular, hydraulic signals—pressure changes in xylem vessels that can propagate at the speed of water flow (Schopfer & Brennicke, 2016; Kuznetsov, 2006).

With a sharp change in water potential, for example, during soil drying, a hydraulic impulse arises in the xylem. This signal can reach leaves within seconds, causing rapid stomatal closure—even before ABA from the roots has time to reach the leaves.

In addition, plants possess electrical signals—changes in membrane potential that propagate through tissues, similar to nerve impulses in animals, but significantly slower (Schopfer & Brennicke, 2016). These signals have been particularly well studied in the sensitive plant (Mimosa pudica) and Venus flytrap (Dionaea muscipula), but they have also been found in many other plants.

Electrical signals can activate calcium channels, causing a Ca²⁺ spike, and thereby trigger the entire cascade of systemic responses. Thus, we see a complex multimodal system of rapid signaling, where hydraulic, electrical, and chemical (ROS, Ca²⁺) signals work in close interconnection.

5.6. Systemic acquired resistance (SAR) as a model of systemic physiology

Now let us see how all these mechanisms work in one of the most studied examples of systemic response—systemic acquired resistance (SAR) (Medvedev, 2012; Taiz et al., 2023).

When a plant encounters a pathogen, a hypersensitive response develops at the infection site—local cell death along with the pathogen. But the defense does not end there. After a few days, the entire plant becomes more resistant to a broad spectrum of pathogens. How does this happen?

The first stage—local reaction. At the infection site:

• NADPH oxidase is activated, generating superoxide and H₂O₂.
• An oxidative burst occurs, killing the pathogen and triggering programmed death of infected cells.
• Synthesis of salicylic acid (SA) is activated.
• Expression of defense genes, including PR proteins and phytoalexin synthesis enzymes, is triggered.

The second stage—generation of the systemic signal. Salicylic acid synthesized at the infection site is partially converted to methyl salicylate—a volatile compound that can spread through the air. In addition, SA and its methyl ester are transported via the phloem to distal parts of the plant (Medvedev, 2012).

The third stage—systemic preparation. In distal tissues:

• Salicylic acid accumulates.
• Expression of PR genes is activated—but not immediately at full capacity, but to a certain "pre-set" level.
• The plant becomes "sensitized"—ready for a faster and stronger response upon repeated attack.

This state can persist for several weeks. Upon reinfection, even a weak signal triggers a powerful defensive response, significantly faster and more effective than during the primary infection.

5.7. SAR as an example of integration of all signaling levels

What makes SAR such a demonstrative model? That it involves all the mechanisms we have discussed throughout the lecture:

1. Calcium—participates in early signaling upon pathogen recognition.

2. ROS—act as signaling molecules triggering the hypersensitive response and activating systemic signals.

3. MAPK cascades—amplify and transduce the signal, activating transcription factors.

4. Hormones (SA, JA, ethylene) —serve as integrators and long-distance signals, distributing roles among different parts of the plant.

5. Systemic waves (ROS, Ca²⁺) —provide rapid alerting of distal tissues.

Thus, SAR is the quintessence of systemic physiology: a local event (infection) through a cascade of signaling events transforms into a global restructuring of the entire organism.

5.8. Mechanisms of stress "memory"

One of the most intriguing features of the systemic response is the plant's ability to "remember" stress experienced. We have already mentioned that SAR can persist for several weeks. But there are other forms of stress memory.

For example, plants that have already experienced drought respond faster and more effectively upon repeated exposure. This phenomenon is called priming (Taiz et al., 2023; Pessarakli, 2020).

Mechanisms of priming may include:

• Epigenetic changes—histone modifications and DNA methylation that alter gene accessibility for transcription. These changes can persist for a long time and even be transmitted to the next generation (Taiz et al., 2023; Lambers & Oliveira, 2019).
• Accumulation of protective proteins and metabolites—for example, osmolytes or heat shock proteins that remain in cells after stress.
• Changes in signaling networks—for example, increased levels of certain transcription factors that make the cell more sensitive to repeated signals.

Thus, the plant does not just respond to stress—it learns from its experience. And this experience, recorded at different levels—from rapid signals to epigenetic memory—becomes part of the survival strategy.

5.9. Systemic physiology as the basis for understanding plant integrity

Colleagues, let us now return to the main question with which we began: how does a local injury transform into a response of the whole plant?

We can now answer it as follows:

1. Local perception—cells at the injury site recognize stress through membrane and intracellular sensors.

2. Intracellular signaling—a calcium signal is triggered, a ROS burst occurs, MAPK cascades are activated.

3. Local reaction—defense programs are launched at the injury site: antioxidant production, hormone synthesis, changes in gene expression.

4. Generation of systemic signal—at the injury site, signals capable of spreading are generated: the ROS wave, the calcium wave, hormonal signals (ABA, SA, JA, ethylene), hydraulic and electrical signals.

5. Propagation of the systemic signal—signals spread throughout the plant: ROS and calcium—through the apoplast and plasmodesmata, hormones—via phloem and xylem, hydraulic and electrical signals—through tissues.

6. Systemic preparation—distal tissues receive the signal and launch "readiness" programs, often at a suboptimal level (priming).

7. Systemic response—upon repeated stress, prepared tissues respond faster and more effectively, ensuring the survival of the whole organism.

5.10. Final summary: the unified organism as a result of signal integration

Colleagues, allow me to summarize our entire lecture. We began with how an individual cell can perceive stress through universal signals—calcium and ROS. Then we saw how these signals are amplified through MAPK cascades and transformed into changes in the genetic program. Next, we understood how hormones integrate these signals at the level of the whole organism, distributing roles among different tissues. And finally, we have seen that all these mechanisms work in a single network, providing a systemic response that turns the plant into a unified, coordinated organism.

What then is a plant in light of all this? It is not just a collection of cells, tissues, and organs. It is a complex communication network, where every cell constantly exchanges information with others, where local events grow into global reorganizations, where past experience determines future responses.

And it is this systemic nature that is the key to understanding stress physiology. Without it, we would see only individual molecules, individual reactions, individual genes. But the plant is more than the sum of its parts. And systemic physiology gives us the tools to understand this wholeness.