Model stress factors

Calcium (Ca²⁺) as a second messenger

An increase in cytosolic Ca²⁺ is one of the earliest events under stress. Calcium ions bind to calcium‑binding proteins—calmodulin and calcineurin‑B‑like proteins (CBL). These proteins, in turn, activate protein kinases, including Ca²⁺‑dependent protein kinases (CDPK) and CBL‑interacting protein kinases (CIPK) (Kuznetsov & Dmitrieva, 2006). Kinase activation triggers a phosphorylation cascade of other target proteins.

Abscisic acid (ABA) as the main hormonal signal

Abscisic acid (ABA) is the key phytohormone mediating the plant's response to water deficit (Schopfer & Brennicke, 2016; Hopkins & Hüner, 2009). Interestingly, ABA is synthesised both in roots and in leaves. Under drought conditions:

1. ABA synthesis is activated in roots;

2. ABA is transported via the xylem to the leaves;

3. Simultaneously, ABA synthesis is enhanced in the leaves themselves.

ABA synthesis from carotenoid precursors (violaxanthin) is catalysed by the enzyme NCED (9‑cis‑epoxycarotenoid dioxygenase). The activity of this enzyme increases sharply when water potential decreases.

In addition, the pH of xylem sap influences signal propagation. During drought, the pH of xylem sap can rise, which promotes the release of ABA from apoplastic stores (Lambers & Oliveira, 2019).

MAPK cascades

Mitogen‑activated protein kinases (MAPKs) are universal components of signalling pathways in eukaryotes. Activation of MAPK cascades is observed under water deficit and leads to phosphorylation of transcription factors (Kosová et al., 2020).

Stomatal closure

This is the fastest and most effective protective reaction (Schopfer & Brennicke, 2016; Hopkins & Hüner, 2009). Under the influence of ABA in guard cells:

1. Efflux of K⁺ and Cl⁻ anions occurs;

2. Cytosolic Ca²⁺ concentration rises;

3. The membrane depolarises;

4. Guard cell turgor decreases;

5. The stomatal pore closes.

Stomatal closure sharply limits transpiration, reducing water loss, but at the same time reduces CO₂ uptake into the leaf.

Growth inhibition

Decreased turgor and altered hormone balance (increase in ABA, decrease in auxins and cytokinins) lead to inhibition of cell expansion (Kuznetsov & Dmitrieva, 2006). Leaf and stem growth slows or stops—this is an adaptive response aimed at reducing the evaporative surface.

Osmotic regulation

Cells accumulate compatible osmolytes—low‑molecular‑weight organic substances that lower the cell water potential without disrupting metabolism (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016). These include:

• Proline—one of the most important amino acids involved in osmoregulation;
• Glycine betaine—an effective osmoprotectant;
• Sugars (sucrose, fructans) and sugar alcohols (sorbitol, mannitol).

Osmolyte synthesis is activated at the transcriptional level. For example, the key enzyme of proline synthesis—pyrroline‑5‑carboxylate synthetase (P5CS)—is induced under drought (Sharma & Dubey, 2020).

Antioxidant defence

When stomata close, CO₂ entry into chloroplasts decreases, leading to over‑reduction of the photosynthetic electron transport chain and formation of reactive oxygen species (ROS). Antioxidant enzymes are activated: superoxide dismutase (SOD), catalase, ascorbate peroxidase, as well as non‑enzymatic antioxidants—ascorbate, glutathione, tocopherols (Medvedev, 2012; Schopfer & Brennicke, 2016).

Synthesis of stress proteins

In response to drought, the expression of genes encoding LEA proteins (Late Embryogenesis Abundant) and dehydrins is activated (Sharma & Dubey, 2020; Medvedev, 2012). These proteins are highly hydrophilic and perform protective functions:

• Stabilise membranes and proteins;
• Bind and retain water;
• Prevent aggregation of denatured proteins.

Changes in root‑to‑shoot ratio

Under prolonged drought, root growth may continue while shoot growth is inhibited. The root‑to‑shoot ratio increases—this allows the plant to increase the absorbing surface in search of water (Kuznetsov & Dmitrieva, 2006).

Protein denaturation

Upon heating, proteins begin to unfold—lose their native conformation. This leads to:

• Loss of enzymatic activity;
• Exposure of hydrophobic patches that are normally buried inside the globule;
• Aggregation—clumping of denatured molecules via hydrophobic contacts.

Particularly sensitive to thermal denaturation are photosynthetic enzymes, primarily RuBisCO (the key enzyme of the C3 cycle) and RuBisCO activase, which is required for its activation (Schopfer & Brennicke, 2016; Kuznetsov & Dmitrieva, 2006). Already at 35–40 °C, the activity of these enzymes begins to decline.

Damage to Photosystem II

One of the most sensitive targets of heat stress is Photosystem II (PSII)—the oxygen‑evolving complex located in the thylakoid membranes of chloroplasts (Nakamoto & Akter, 2020; Schopfer & Brennicke, 2016). The first to be damaged is the oxygen‑evolving complex (OEC)—the protein‑manganese cluster that catalyses water splitting. Upon heating to 40–45 °C, this complex is inactivated, leading to:

• Disruption of electron transfer from water to P680;
• Accumulation of reduced components of the electron transport chain;
• Formation of reactive oxygen species (ROS).

The PSII reaction centre also suffers, especially the D1 protein, which has the highest turnover among all plant proteins and constantly requires repair (Hopkins & Hüner, 2009).

Changes in membrane fluidity

At elevated temperatures, membrane lipids become more fluid. This disrupts:

• Function of membrane proteins—channels, carriers, receptors;
• Proton conductance of membranes;
• Coupling of electron transport with photophosphorylation and oxidative phosphorylation (Schopfer & Brennicke, 2016).

As a result, ATP synthesis efficiency declines, and the cell experiences energy deficit.

Water deficit as a secondary factor

At high temperatures, transpiration increases sharply. If water uptake from the soil is limited, this leads to the development of water deficit—so‑called heat drought (Kuznetsov & Dmitrieva, 2006). Thus, heat stress can trigger a cascade of secondary disturbances that amplify damage.

Membrane fluidity as a primary sensor

According to current views, primary heat perception is linked to changes in membrane fluidity (Schopfer & Brennicke, 2016). Membrane lipids become more mobile upon heating. This change in the physical state of the membrane affects the conformation of embedded receptor proteins.

It is thought that receptor‑like kinases and two‑component histidine kinases act as thermal sensors, capable of changing their activity depending on the physical state of the lipid bilayer. In cyanobacteria, the histidine kinase Hik33, sensitive to changes in membrane fluidity upon heating, has been identified (Schopfer & Brennicke, 2016). In higher plants, similar mechanisms are currently being actively investigated.

Protein denaturation as a signal

Partially denatured proteins exposing hydrophobic patches serve as another signal. These "abnormal" proteins are recognised by cellular protein quality control systems, triggering signalling cascades and activation of protective programmes (Nakamoto & Akter, 2020).

Calcium signal

As with other stresses, heat stress causes a rapid influx of Ca²⁺ into the cytosol (Schopfer & Brennicke, 2016). Calcium acts as a universal second messenger, activating calcium‑dependent protein kinases and triggering phosphorylation cascades.

Heat shock factors (HSF)

Plants possess a large family of HSFs (19 to 52 genes in different species), divided into classes A, B, and C (Nakamoto & Akter, 2020; Schopfer & Brennicke, 2016). Class A1 HSFs play a key role in the primary response—they are the "master regulators" of the heat shock response.

Mechanism of HSF activation (Schopfer & Brennicke, 2016; Kuznetsov & Dmitrieva, 2006):

1. At normal temperature, HSF exists in the cytoplasm as an inactive monomer bound to molecular chaperones (primarily Hsp70 and Hsp90).

2. Upon temperature increase, these chaperones are "distracted" by binding to denatured proteins.

3. HSF is released, forms a homotrimer, and translocates to the nucleus.

4. In the nucleus, the HSF trimer binds to the heat shock element (HSE) in the promoter regions of HSP genes.

5. Transcription of HSP genes is initiated, and HSP mRNA can be detected within 3–5 minutes after the onset of heating (Nakamoto & Akter, 2020; Kuznetsov & Dmitrieva, 2006).

Importantly, heat shock genes are intron‑less, meaning they do not require splicing. This ensures maximum speed of their activation—a critical condition for survival during rapid temperature increase (Kuznetsov & Dmitrieva, 2006).

Calcium‑dependent pathways

In parallel with the HSF pathway, calcium signals and MAPK cascades are activated (Kosová et al., 2020; Schopfer & Brennicke, 2016). They modulate the activity of HSFs and other transcription factors.

ABA and other phytohormones

Under heat stress, ABA content may also increase, although its role here is less pronounced than under drought (Kuznetsov & Dmitrieva, 2006). ABA promotes stomatal closure, limiting water loss. Ethylene synthesis also increases, which participates in the regulation of defence responses.

Synthesis of heat shock proteins (HSPs)

This is the central element of the heat response. HSPs are molecular chaperones that perform protective functions (Nakamoto & Akter, 2020; Schopfer & Brennicke, 2016).

Main HSP families in plants:

Particularly interesting are small HSPs—they are ATP‑independent holdases that create temporary depots for denatured proteins, preventing their aggregation. These proteins are then handed over to ATP‑dependent chaperone systems (Hsp70 and Hsp100) for refolding or degradation (Nakamoto & Akter, 2020).

It is important to note that not all HSPs are heat‑inducible. Some of them (e.g., Hsp70, Hsp90) are present constitutively—they are called heat shock cognate proteins (Hsc) and participate in normal protein folding, but their synthesis is also enhanced under stress (Kosová et al., 2020).

Changes in membrane lipid composition

Under prolonged exposure to elevated temperatures, membrane adaptation occurs: the content of saturated fatty acids increases, which reduces excessive membrane fluidity and increases their thermal stability (Schopfer & Brennicke, 2016). This process requires time and synthesis of new molecules.

Enhanced transpiration

Under short‑term heat, the plant may increase transpiration for leaf cooling. This is an effective mechanism, but it works only when water supply from the soil is sufficient. Under combined heat and drought, transpiration is limited by stomatal closure, and cooling becomes impossible.

Antioxidant defence

Under heat stress, as under other stresses, the formation of reactive oxygen species (ROS) increases, especially in chloroplasts due to PSII damage. Enzymatic and non‑enzymatic antioxidant systems are activated (Schopfer & Brennicke, 2016; Medvedev, 2012). Synthesis of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase is enhanced.

Inhibition of most protein synthesis

Under heat shock, synthesis of most "normal" proteins is sharply suppressed—this is a translational block. This block allows the cell to redirect resources to HSP synthesis and avoid synthesising proteins that could be damaged or assembled incorrectly at high temperature (Schopfer & Brennicke, 2016).

Acquisition of thermotolerance

Short‑term exposure to moderately high temperature (e.g., 38 °C for 30–60 minutes) makes the plant more resistant to critically high temperature (45–50 °C), which would normally be lethal. This phenomenon is called acquired thermotolerance (Nakamoto & Akter, 2020; Schopfer & Brennicke, 2016). The level of thermotolerance positively correlates with the amount of accumulated HSPs and their persistence.

Recovery

After relief of heat stress (return to optimal temperature):

1. HSP synthesis ceases—within minutes, HSP mRNA levels drop sharply.

2. Already synthesised HSPs are gradually degraded with half‑lives ranging from hours to days.

3. Normal protein synthesis resumes.

4. Damaged proteins are either refolded (with the help of HSPs) or targeted for proteasomal degradation (with the participation of ubiquitin, also induced by heat) (Nakamoto & Akter, 2020).

Importantly, short‑term heat stress may leave no visible damage if HSPs have had time to perform their protective function.

Acclimation (long‑term adaptation)

Under gradual or repeated exposure to elevated temperatures, the plant can acclimatise to heat. This manifests as:

1. Shift in the temperature optimum of photosynthesis towards higher temperatures. For example, in oleander grown at 20 °C and 45 °C, the temperature curves of photosynthesis shift accordingly, so that the optimum temperature matches the growth temperature (Schopfer & Brennicke, 2016).

2. Changes in thermal stability of enzymes. Thermostable isoforms of enzymes appear; in cereals, a thermostable isoform of RuBisCO activase is induced, replacing the thermolabile one at high temperatures (Schopfer & Brennicke, 2016).

3. Accumulation of "reserve" HSPs. In acclimated plants, the basal level of some HSPs may be elevated.

4. Membrane lipid adaptation.

Damage

At too high a temperature (usually >45–50 °C for most plants) or with too rapid heating (when HSPs do not have time to be synthesised), irreversible damage occurs:

1. Irreversible protein denaturation and aggregation;

2. Membrane destruction (excessive fluidity, loss of barrier function, ion leakage);

3. Inactivation of PSII and other key enzymes;

4. Uncoupling of oxidative phosphorylation, cessation of ATP synthesis;

5. Tissue necrosis and plant death (Kuznetsov & Dmitrieva, 2006).

Cost of adaptation under heat stress: even successful acclimation requires energy expenditure for HSP synthesis and metabolic reorganisation, which reduces growth rate, productivity, and yield (Schopfer & Brennicke, 2016).

Special case: freezing

At negative temperatures, membrane disturbances are compounded by ice formation. Initially, ice forms extracellularly—in intercellular spaces and xylem vessels. This creates a very low water potential in the extracellular space, and water begins to leave the cells along the gradient. Cells dehydrate—freezing‑induced dehydration develops (Schopfer & Brennicke, 2016; Li, 1994).

If cooling occurs slowly (as in nature in winter), cells have time to adapt to this dehydration. If rapid, ice forms inside cells, leading to mechanical disruption of membranes and cell death (Li, 1994; Schopfer & Brennicke, 2016).

Thus, under freezing stress, the primary disturbance is ice formation, and the secondary one is dehydration, similar to drought stress.

Membrane sensors

It is believed that the primary cold sensors are two‑component histidine kinases and receptor‑like kinases embedded in the plasma membrane and endomembranes (Schopfer & Brennicke, 2016; Kosová et al., 2020). In cyanobacteria, the best‑studied protein is Hik33, which senses changes in membrane fluidity. In higher plants, the protein COLD1 has been identified—it interacts with a G‑protein and is involved in cold perception in rice. Mutants in COLD1 lose the ability for cold acclimation (Kosová et al., 2020).

Mechanosensitive channels

Changes in membrane fluidity can activate mechanosensitive Ca²⁺ channels, leading to a rapid influx of calcium into the cytosol (Schopfer & Brennicke, 2016). Within seconds after cooling, a calcium wave can be detected.

Perception through changes in redox state

Upon cooling, the operation of electron transport chains in chloroplasts and mitochondria is disturbed, changing the cellular redox state. This can also serve as a signal perceived by redox‑sensitive proteins (Hopkins & Hüner, 2009).

Calcium signal and MAPK cascades

The influx of Ca²⁺ activates calcium‑binding proteins (calmodulin, CBL) and calcium‑dependent protein kinases (CDPK, CIPK). This triggers MAPK cascades and phosphorylation of transcription factors (Kosová et al., 2020; Medvedev, 2012).

ABA‑dependent pathway

Under cold, as under drought, the content of abscisic acid (ABA) may increase. ABA activates transcription factors with bZIP domains (AREB/ABF), which bind to ABRE elements in the promoters of cold‑regulated genes (Kosová et al., 2020; Li, 1994).

ABA‑independent pathway—the CBF/DREB system

This is the best‑studied and probably the main pathway for cold acclimation in temperate plants (Schopfer & Brennicke, 2016; Kosová et al., 2020; Sharma & Dubey, 2020).

1. Upon cooling (usually at +4…+10 °C), genes encoding transcription factors CBF1, CBF2, CBF3 (C‑repeat Binding Factor, also called DREB1) are activated.

2. CBF proteins bind to the CRT/DRE element (CCGAC) in the promoters of so‑called COR genes (cold‑regulated).

3. Expression of dozens of COR genes is triggered, whose products participate in cold protection.

Important: the CBF pathway is induced not by ABA but directly by cold. However, there is cross‑regulation—some genes are activated through both CBF and ABA.

Changes in fatty acid composition of membranes (desaturation)

Upon temperature decrease, desaturase enzymes are activated, which introduce double bonds into the fatty acid chains of membrane lipids (Li, 1994; Medvedev, 2012). This increases the proportion of unsaturated fatty acids, lowers the phase transition temperature, and restores membrane fluidity at low temperatures.

In cold‑tolerant plants, this process may take several days, but it is critically important for long‑term adaptation. Interestingly, in some species, desaturation is induced only during slow cooling (hardening) and does not occur upon rapid cooling.

Synthesis of cold‑regulated proteins (COR proteins)

COR proteins are a group of proteins encoded by COR genes (Kosová et al., 2020; Sharma & Dubey, 2020; Medvedev, 2012). These include:

1. LEA proteins and dehydrins—highly hydrophilic proteins that protect other proteins and membranes from dehydration damage. They contain many repeated glycine‑, alanine‑, and proline‑rich segments and can tightly bind water (Medvedev, 2012).

2. Antifreeze proteins (AFPs)—proteins that bind to ice crystals and inhibit their growth (Li, 1994; Schopfer & Brennicke, 2016). They accumulate in the apoplast, where extracellular ice formation occurs. Interestingly, many AFPs share structural similarity with pathogenesis‑related (PR) proteins—for example, chitinases and β‑1,3‑glucanases (Schopfer & Brennicke, 2016).

3. Other stress proteins—chaperones (including some HSPs), antioxidant proteins, enzymes of osmolyte metabolism.

Accumulation of osmolytes and sugars

Under cold, as under drought, compatible osmolytes accumulate in cells, especially sucrose, raffinose, fructans, as well as proline and glycine betaine (Li, 1994; Schopfer & Brennicke, 2016; Medvedev, 2012). Sugars have a dual role:

• Bind water, reducing the amount of free water available for ice formation;
• Stabilise membranes and proteins, acting as cryoprotectants.

In winter cereals, sucrose content in tissues can increase from 2–3% to 20–25% of dry mass during hardening (Kuznetsov & Dmitrieva, 2006).

Changes in hormone balance

Under cold, ABA and ethylene content increase, while auxin and gibberellin content decrease. This leads to growth inhibition—an adaptive response that redirects resources to hardening (Li, 1994; Medvedev, 2012).

Antioxidant defence

At low temperatures, especially in combination with high light (e.g., winter clear days), ROS formation increases. Superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase are activated (Schopfer & Brennicke, 2016; Medvedev, 2012).

Special mechanisms under freezing

At negative temperatures, additional mechanisms are engaged:

1. Controlled extracellular dehydration. Cells lose water to the intercellular spaces, where it freezes. This allows avoidance of intracellular ice (Schopfer & Brennicke, 2016).

2. Supercooling. Water in cells remains liquid at temperatures significantly below 0 °C (down to –40 °C in some woody species). This is achieved through high osmolyte concentration and absence of nucleation centres (Li, 1994; Schopfer & Brennicke, 2016).

3. Vitrification (glass transition). At very low temperatures (below –40 °C), the cytoplasm can enter a glassy state—extremely viscous but not crystalline. This allows cells to survive extreme frost without ice formation (Schopfer & Brennicke, 2016).

4. Suppression of intracellular ice. Plants prevent ice formation inside cells by maintaining high osmotic pressure and using antifreeze proteins and sugars.

Recovery

Upon return to optimal temperatures:

• Synthesis of COR proteins, desaturases, and antifreeze proteins ceases;
• Accumulated osmolytes are gradually consumed or incorporated into metabolism;
• Normal membrane composition is restored, but this may take several days (desaturation is reversible);
• Growth and photosynthesis resume.

In heat‑loving plants, brief cooling (e.g., night frosts) may allow rapid repair if the cooling did not cause irreversible damage.

Acclimation (cold hardening)

This is a key property of winter‑hardy plants. Cold hardening is a process that occurs in autumn with gradual temperature decrease and day shortening, and consists of two phases (Li, 1994; Schopfer & Brennicke, 2016):

First phase (photoperiodic)—induced by short days, controlled by phytochrome. The plant enters dormancy, growth ceases. Frost resistance increases by 5–10 °C.

Second phase (actual low‑temperature)—induced by exposure to low positive temperatures (0…+5 °C). Expression of COR genes (CBF pathway) is triggered, sugars, dehydrins, antifreeze proteins accumulate, and membrane fatty acid composition changes. Frost resistance increases to the maximum level (e.g., in winter wheat from –5…–7 to –30…–35 °C) (Kuznetsov & Dmitrieva, 2006).

Important: hardening requires time and energy—slow cooling gives the plant time to prepare. Rapid cooling does not harden but damages.

Hardening and drought resistance. There is cross‑protection between cold and drought acclimation—many mechanisms are shared: accumulation of osmolytes, LEA proteins, antioxidants. Therefore, plants hardened to drought often become more frost‑resistant (Schopfer & Brennicke, 2016; Li, 1994).

Damage

Under too severe or rapid cooling (or in the absence of hardening capacity, as in heat‑loving plants), irreversible damage occurs:

Under chilling stress (positive temperatures):

• Irreversible disruption of membrane structure (gel state, loss of semipermeability);
• Leakage of ions and metabolites;
• Inactivation of enzymes;
• Disruption of photosynthesis and respiration;
• Tissue necrosis (especially in heat‑loving species—maize, tomato, cucumber) (Medvedev, 2012; Kuznetsov & Dmitrieva, 2006).

Under freezing:

• Intracellular ice formation—mechanical destruction of organelle and plasma membranes;
• Irreversible dehydration—water loss leads to protein denaturation and membrane destruction, even if ice is extracellular (Schopfer & Brennicke, 2016);
• Damage upon thawing—during rapid thawing, water from melting crystals can cause osmotic shock and cell rupture.

Osmotic phase (rapid, first hours–days)

High salt concentration (primarily NaCl) in the soil solution lowers its water potential. By osmotic laws, water moves towards the lower water potential. If the soil solution has a lower Ψ_w than root cells, water begins to leave the roots into the soil (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016).

The primary disturbance is osmotic shock and loss of turgor. In terms of physiological effect, the osmotic phase of salinity is almost indistinguishable from drought: drop in water potential, stomatal closure, growth inhibition, osmolyte accumulation (Kuznetsov & Dmitrieva, 2006).

However, unlike drought, where the problem is solved by restoring water supply, under salinity the osmotic phase merely precedes a more serious problem—ion toxicity.

Ionic phase (slow, days–weeks)

If the plant continues to be exposed to saline conditions, along with water, Na⁺ and Cl⁻ ions begin to enter the roots. Unlike K⁺, Ca²⁺, and other macronutrients, sodium is not essential for most plants and does not perform specific biochemical functions (Lambers & Oliveira, 2019).

Accumulation of Na⁺ in the cytoplasm causes:

• Ion imbalance—disruption of the K⁺/Na⁺ ratio, critically important for the function of many enzymes;
• Toxicity—Na⁺ ions compete with K⁺ for binding sites on enzymes, leading to their inactivation;
• Disruption of protein synthesis—at high Na⁺ concentrations, the translation apparatus is affected (Sharma & Dubey, 2020);
• Disruption of photosynthesis—damage to chloroplasts and inhibition of enzymes (Kuznetsov & Dmitrieva, 2006).

Cl⁻ is also toxic, but its damaging effect is usually less pronounced than that of Na⁺ and varies more among species.

Thus, under salinity, both water status and ion homeostasis are disrupted simultaneously, and each of these factors requires its own adaptive mechanisms (Munns & Tester, 2008).

Perception of the osmotic phase

Similar to drought—via osmosensors and mechanosensitive channels. The drop in turgor and changes in membrane tension activate Ca²⁺ channels, causing calcium influx into the cytosol (Kosová et al., 2020; Schopfer & Brennicke, 2016). This signal triggers ABA synthesis (both in roots and leaves) and osmotic adaptation.

Perception of the ionic phase—the SOS system

The best‑studied mechanism for sensing excess Na⁺ is the SOS system (Salt Overly Sensitive), identified in Arabidopsis (Medvedev, 2012; Kosová et al., 2020; Kuznetsov & Dmitrieva, 2006).

The system includes three key proteins:

1. SOS3—a calcium‑sensor protein containing three EF‑hands for Ca²⁺ binding. When cytosolic Na⁺ concentration rises, Ca²⁺ channels open, calcium binds to SOS3, causing conformational changes.

2. SOS2—a Ca²⁺‑dependent protein kinase (CIPK24). Activated SOS3 recruits SOS2 to the plasma membrane and activates it. SOS2 phosphorylates its target protein.

3. SOS1—a plasma membrane Na⁺/H⁺ antiporter. Phosphorylation of SOS1 activates it—it begins to pump Na⁺ out of the cell into the apoplast, using the energy of the proton gradient generated by the H⁺‑ATPase.

Physiological significance: the plant "senses" the rise in Na⁺ in the cytoplasm via the calcium signal and activates active sodium extrusion from the cell—the first level of defence against ion toxicity (Medvedev, 2012).

Other sensory mechanisms

Besides the SOS system, other mechanisms may participate in salinity perception:

• Receptor‑like kinases (RLK) sensitive to changes in ion composition;
• Aquaporins changing activity in response to osmotic shock;
• Changes in membrane potential—Na⁺ influx depolarises the membrane, which can serve as an additional signal (Lambers & Oliveira, 2019).

Calcium signal and MAPK cascades

The Ca²⁺ wave initiated by osmotic and ionic stress activates calcium‑dependent protein kinases (CDPK) and the CBL‑CIPK system (including the SOS pathway). This, in turn, triggers MAPK cascades and phosphorylation of transcription factors (Kosová et al., 2020).

ABA‑dependent pathway

Under salinity, as under drought, ABA synthesis increases (Lambers & Oliveira, 2019; Schopfer & Brennicke, 2016). ABA:

• Induces stomatal closure to limit transpiration and salt entry with the water flow;
• Activates expression of osmolyte genes;
• Promotes accumulation of LEA proteins and dehydrins (Sharma & Dubey, 2020).

ABA‑independent pathway

A number of salt‑regulated genes are activated without ABA involvement. In particular, genes encoding components of the SOS system (SOS1, SOS2, SOS3) are induced directly by Na⁺ via the calcium signal (Medvedev, 2012).

Systemic signals

Under salinity, roots generate signals transmitted to shoots:

• ABA is transported via the xylem;
• Xylem sap pH may change, affecting ABA distribution among compartments;
• Ionic signals—changes in K⁺, Ca²⁺, Na⁺ concentrations in xylem sap (Lambers & Oliveira, 2019).

Strategy 1: Ion compartmentation into the vacuole

This is the main strategy for protecting the cytoplasm from toxic Na⁺ concentrations (Schopfer & Brennicke, 2016; Lambers & Oliveira, 2019; Kuznetsov & Dmitrieva, 2006).

On the tonoplast (vacuolar membrane), a vacuolar Na⁺/H⁺ antiporter (NHX1) operates. It pumps Na⁺ into the vacuole using the energy of the proton gradient generated by:

• V‑ATPase (vacuolar ATPase);
• V‑PPase (vacuolar pyrophosphatase).

As a result, high Na⁺ concentrations (up to 200–500 mM) accumulate in the vacuole, while cytoplasmic levels remain relatively low (10–50 mM). To osmotically balance Na⁺ accumulation in the vacuole, compatible osmolytes are synthesised in the cytoplasm (see below).

In halophytes (plants adapted to salinity), this mechanism is particularly efficient—they can accumulate up to 1 M NaCl or more in vacuoles (Kuznetsov & Dmitrieva, 2006).

Strategy 2: Active ion extrusion from the cell

In parallel with vacuolar compartmentation, SOS1—a plasma membrane Na⁺/H⁺ antiporter—pumps Na⁺ out of the cell into the apoplast (Medvedev, 2012). This is effective for roots, where the apoplast is the soil solution or cell walls.

The combination of vacuolar accumulation and plasma membrane extrusion allows cytosolic Na⁺ concentration to be maintained at a safe level.

Strategy 3: Synthesis of compatible osmolytes

As under drought, under salinity, compatible osmolytes accumulate in the cytoplasm (Schopfer & Brennicke, 2016; Lambers & Oliveira, 2019; Sharma & Dubey, 2020):

• Glycine betaine—the most effective osmoprotectant, especially in halophytes of the family Chenopodiaceae (beet, goosefoot, saltworts) (Kuznetsov & Dmitrieva, 2006);
• Proline—a universal osmolyte synthesised by many plants;
• Sugars—sucrose, fructans, as well as sugar alcohols (sorbitol, mannitol) and cyclic polyols (pinitol).

Osmolyte synthesis is activated at the transcriptional level—genes encoding biosynthetic enzymes (e.g., P5CS for proline, BADH for glycine betaine) are induced. Osmolyte accumulation serves two functions:

• Osmotic—lowering cytoplasmic water potential to balance vacuolar pressure;
• Protective—stabilising proteins and membranes under osmotic and ionic stress.

Strategy 4: Structural protection—salt glands and salt bladders

Halophytes possess specialised structures for removing excess salt from the organism (Kuznetsov & Dmitrieva, 2006; Schopfer & Brennicke, 2016):

1. Salt glands—multicellular structures that actively secrete NaCl onto the leaf surface (e.g., in tamarisk, some grasses). Salt crystallises on the surface and is later washed off by rain or blown away.

2. Salt bladders (bladder hairs)—specialised trichomes that accumulate salt (e.g., in some Chenopodiaceae—goosefoot, saltwort). When fully filled, the bladder dies and falls off, removing salt from the plant.

3. Salt secretion by roots—some plants can excrete excess salt back into the soil through roots.

Strategy 5: Limiting ion transport to shoots

Roots regulate ion entry into the xylem:

• Na⁺ is retained in root cortex cells;
• Na⁺ can be re‑excreted from the xylem back into the cortex (or into the soil);
• Minimal Na⁺ enters the xylem vessels (Lambers & Oliveira, 2019; Kuznetsov & Dmitrieva, 2006).

In some plants, salts accumulate in old leaves, which then abscise—an additional mechanism for removing toxic ions.

Strategy 6: Stomatal closure

As under drought, under salinity, stomata close under the influence of ABA. This:

• Limits transpiration and water loss;
• Reduces salt entry with the water flow into shoots (Lambers & Oliveira, 2019).

However, stomatal closure simultaneously reduces CO₂ uptake and photosynthesis—this is the "cost" of protection.

Strategy 7: Antioxidant defence

Under salinity, as under other stresses, ROS formation is activated. Enzymatic (SOD, catalase, ascorbate peroxidase) and non‑enzymatic (ascorbate, glutathione, tocopherols) antioxidant systems are engaged (Schopfer & Brennicke, 2016; Medvedev, 2012).

Gene expression regulation

Under salinity, transcription of a wide range of genes is activated, encoding:

• Ion transporters (SOS1, NHX1, HKT1);
• Osmolyte synthesis enzymes (P5CS, BADH);
• LEA proteins and dehydrins;
• Antioxidant enzymes;
• Aquaporins (regulation of water permeability);
• ABA metabolism enzymes (Sharma & Dubey, 2020; Kosová et al., 2020; Medvedev, 2012).

Recovery

If salinisation ceases (e.g., salts leached from soil by irrigation water), the plant can recover:

• Na⁺ and Cl⁻ ions are washed out from tissues or redistributed;
• Osmolyte and stress protein synthesis ceases;
• Normal ion ratios are restored;
• Growth and photosynthesis resume.

However, full recovery may take several days, especially after severe ionic stress.

Acclimation (increase in salt tolerance)

Under prolonged or repeated salinisation, the plant can acclimate:

• Efficiency of the SOS system and NHX transporters increases;
• Basal level of osmolytes (proline, glycine betaine) rises;
• Membrane lipid composition changes (increase in unsaturated fatty acids);
• Antioxidant defence is activated;
• Leaves develop xeromorphic traits—thicker cuticle, smaller cells (Kuznetsov & Dmitrieva, 2006).

In some halophytes, acclimation can lead to induction of CAM photosynthesis—a shift from C3 to crassulacean acid metabolism, allowing water conservation under osmotic stress (Schopfer & Brennicke, 2016).

Damage

At too high salt concentrations (or in the absence of genetic adaptation capacity), irreversible damage occurs:

1. Irreversible ion imbalance—disruption of K⁺/Na⁺ ratio leads to inactivation of key enzymes (Kuznetsov & Dmitrieva, 2006);

2. Protein degradation—high Na⁺ concentrations cause protein denaturation and aggregation (Sharma & Dubey, 2020);

3. Oxidative damage—lipid peroxidation of membranes, damage to chloroplasts and mitochondria;

4. Tissue necrosis—especially in old leaves where salts accumulate;

5. Plant death.

Cost of adaptation under salinity is particularly high: energy spent on osmolyte synthesis, ion pump operation, and stress proteins reduces productivity. Even moderate salinity in glycophytes can lead to significant yield losses (Munns & Tester, 2008; Lambers & Oliveira, 2019).

Oxygen deficit in the root zone

When soil is flooded, water fills the pore space, displacing air. The diffusion coefficient of oxygen in water is about 10⁴ times lower than in air. Plant roots, soil microorganisms, and animals rapidly consume available oxygen, and its concentration in the soil solution drops to critical levels (Schopfer & Brennicke, 2016).

The primary disturbance is cessation of oxygen access to root cell mitochondria. In the absence of the final electron acceptor (O₂), the respiratory electron transport chain stops, and:

1. The Krebs cycle ceases (requires NAD⁺, which is not regenerated);

2. Oxidative phosphorylation is blocked;

3. ATP synthesis in mitochondria stops.

The only source of ATP remains glycolysis, which under anaerobic conditions yields only 2 molecules of ATP per glucose (compared to 36–38 under aerobic respiration) (Medvedev, 2012).

Energy crisis

The decline in ATP level is the most rapid and critical disturbance under hypoxia. Without ATP, the following are impossible:

• Active transport across membranes (H⁺‑ATPases);
• Synthesis of proteins, nucleic acids, lipids;
• Maintenance of ion homeostasis;
• Growth processes.

Accumulation of toxic products—acidosis

Under anaerobic conditions, glycolysis produces pyruvate, which is not oxidised in the Krebs cycle but is reduced. Depending on the type of fermentation:

• Lactic acid fermentation (activated by lactate dehydrogenase, LDH) yields lactic acid.
• Alcoholic fermentation (activated by pyruvate decarboxylase and alcohol dehydrogenase, ADH) yields ethanol.

Accumulation of lactic acid causes acidosis—a drop in cytoplasmic pH from normal 7.4 to 6.8 and below. Acidosis damages enzymes, membranes, and protein synthesis (Medvedev, 2012). Therefore, hypoxia‑tolerant species quickly switch from lactic to alcoholic fermentation—ethanol diffuses out of cells and does not cause acidification.

Thus, under hypoxia, three interrelated parameters are disturbed simultaneously: ATP level, redox balance (NADH/NAD⁺), and cytoplasmic pH.

Haemoglobins as oxygen sensors

Plants possess haemoglobins—haem‑containing proteins capable of binding O₂. They are thought to act as sensors responding to decreased oxygen concentration (Kuznetsov & Dmitrieva, 2006; Medvedev, 2012).

Haemoglobins can:

• Bind O₂ with high affinity;
• Change conformation depending on pO₂;
• Transmit signals through interaction with other proteins.

Changes in redox state of the respiratory chain

Upon arrest of the electron transport chain, reduced carriers (NADH, ubiquinone, cytochromes) accumulate. Changes in the redox status of the cell serve as an important signal perceived by redox‑sensitive proteins that influence gene expression (Schopfer & Brennicke, 2016; Kosová et al., 2020).

Calcium signal

Under anoxia, a rapid influx of Ca²⁺ into the cytosol is observed. Possible calcium sources include mitochondria (where electron transport chain function is disrupted) and the extracellular space through plasma membrane channels (Medvedev, 2012). Within minutes after the onset of anoxia, an increase in [Ca²⁺]_cyt and a rise in alcohol dehydrogenase mRNA levels are recorded (Medvedev, 2012).

Energy status—ATP/ADP ratio

The drop in ATP and rise in ADP/AMP are direct signals of energy crisis. These changes affect regulation of metabolic pathways and gene expression, although the mechanisms of direct transduction of this signal are not yet fully elucidated (Schopfer & Brennicke, 2016).

Calcium signal and MAPK cascades

The Ca²⁺ influx activates calcium‑binding proteins and protein kinases, which in turn trigger MAPK cascades. This leads to phosphorylation of transcription factors regulating the expression of anaerobic response genes (Kosová et al., 2020; Medvedev, 2012).

Ethylene as a systemic signal

Under root hypoxia, synthesis of 1‑aminocyclopropane‑1‑carboxylic acid (ACC)—the immediate precursor of ethylene—is activated (Medvedev, 2012; Schopfer & Brennicke, 2016). ACC is transported via xylem from roots to shoots, where it is converted to ethylene by ACC oxidase (which requires oxygen).

Ethylene, reaching the shoots, causes:

• Epinasty—downward bending of leaves and petioles, characteristic of many species under flooding;
• Induction of aerenchyma formation;
• Other adaptive responses (Schopfer & Brennicke, 2016; Medvedev, 2012).

ABA under hypoxia

Under flooding, ABA content may also increase, causing stomatal closure and reduced transpiration (Kuznetsov & Dmitrieva, 2006). This limits water loss but may also exacerbate the energy crisis.

Switch to anaerobic metabolism

The main metabolic response is the transition from aerobic respiration to glycolysis and fermentation (Medvedev, 2012; Schopfer & Brennicke, 2016).

1. Glycolysis—breakdown of glucose to pyruvate yielding 2 ATP and 2 NADH.

2. Alcoholic fermentation—reduction of pyruvate to ethanol involving pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH):

Pyruvate → acetaldehyde + CO₂ (PDC)

Acetaldehyde + NADH + H⁺ → ethanol + NAD⁺ (ADH)

This reaction regenerates NAD⁺, which is required for glycolysis to continue, and does not acidify the cell.

3. Lactic acid fermentation (in early stages)—reduction of pyruvate to lactate by lactate dehydrogenase (LDH). However, lactate accumulation causes acidosis, so in tolerant species a rapid switch to alcoholic fermentation occurs.

Synthesis of anoxia proteins

Under anoxia, synthesis of most "normal" proteins is suppressed, but synthesis of about 20 specific polypeptides—anoxia proteins—is activated (Kuznetsov & Dmitrieva, 2006; Medvedev, 2012). These are mainly:

• Alcohol dehydrogenase (ADH)—key enzyme of alcoholic fermentation;
• Pyruvate decarboxylase;
• Glucose phosphate isomerase;
• Glyceraldehyde‑3‑phosphate dehydrogenase (glycolytic enzyme);
• Enolase (glycolytic enzyme);
• Sucrose synthase—provides substrates for glycolysis from sucrose;
• α‑Amylase—hydrolyses starch to glucose.

These proteins are considered isoforms of enzymes optimised for anaerobic conditions (increased stability at low pH, high activity at low ATP).

Formation of aerenchyma

Under prolonged hypoxia, tolerant species form aerenchyma—tissue with large intercellular spaces (air cavities) that facilitate oxygen transport from the shoot to the roots (Medvedev, 2012; Schopfer & Brennicke, 2016).

The process involves programmed cell death (apoptosis):

1. Under the influence of ethylene, a death programme is activated in root cortex cells;

2. Cell walls and contents are degraded;

3. Cells collapse, forming cavities;

4. Cavities coalesce into continuous air channels.

Aerenchyma not only serves for oxygen transport but also reduces tissue density, facilitating growth in water.

Root growth towards oxygen

Under hypoxia, roots may exhibit aerotropism—growth towards zones with higher oxygen content. Enhanced root growth deeper into the soil, where aeration may be better, is also observed (Kuznetsov & Dmitrieva, 2006). This process depends on assimilate availability.

Reduction of energy‑consuming processes

To save ATP under energy crisis, the plant:

• Inhibits growth (cell division and expansion);
• Reduces protein and nucleic acid synthesis;
• Decreases activity of pumps and transporters.

Antioxidant defence

Under hypoxia, as under other stresses, ROS formation is activated, especially upon oxygen return (reoxygenation). Antioxidant systems—SOD, catalase, ascorbate peroxidase—are engaged (Medvedev, 2012; Schopfer & Brennicke, 2016).

Adaptations of aquatic plants

In permanently aquatic plants (e.g., water lilies), aerenchyma is constitutive. In many aquatic species, flooding stimulates intense growth of petioles and internodes to bring leaves to the surface for air access. This process is controlled by ethylene and can be very rapid—in some species, daily increments reach several centimetres (Schopfer & Brennicke, 2016).

Recovery

Upon return to normal soil aeration:

• Anaerobic metabolism is switched off;
• Synthesis of ADH and other fermentation enzymes ceases; accumulated proteins degrade;
• Mitochondrial function and oxidative phosphorylation are restored;
• Cytoplasmic pH normalises;
• Growth resumes.

However, recovery may be accompanied by oxidative stress upon sudden oxygen return (reoxygenation)—ROS formation can damage tissues weakened by prolonged hypoxia (Schopfer & Brennicke, 2016; Medvedev, 2012).

Acclimation

In hypoxia‑tolerant species (rice, many aquatic and wetland plants, willow), during prolonged flooding, the following develop:

• Well‑developed aerenchyma;
• Efficient oxygen transport system (including barriers against O₂ leakage from roots into soil);
• High activity of glycolytic and fermentative enzymes;
• Mechanisms to prevent acidosis (switch to alcoholic fermentation, active lactate excretion) (Medvedev, 2012);
• Increased resistance to reoxygenation stress (activation of antioxidant defence).

Example: rice can grow at depths of 50 cm or more thanks to well‑developed aerenchyma and the ability for rapid internode elongation under flooding (Kuznetsov & Dmitrieva, 2006).

Damage

In intolerant species (most mesophytes—maize, bean, tomato, wheat), prolonged hypoxia leads to irreversible damage:

1. Irreversible acidosis—cytoplasmic pH drops below critical levels, denaturing enzymes and membrane proteins;

2. ATP depletion—glycolysis cannot sustain necessary ATP levels; energy starvation leads to structural breakdown;

3. Membrane disruption—loss of barrier function, leakage of ions and metabolites;

4. Root death—especially the absorption zone (root hairs), halting water and mineral uptake (Kuznetsov & Dmitrieva, 2006);

5. Shoot death due to water and mineral deficiency, as well as anaerobic processes in roots.