Vacuolar system and cellular inclusions

Maintenance of shape of non‑lignified organs (leaves, herbaceous stems, petals);

Cell growth by expansion — the basis for increasing cell size is water uptake by the vacuole, not the synthesis of new cytoplasm (Takatsuka et al., 2023);

Resistance to mechanical loads (wind, rain).

Water — the main component of cell sap, mobilized when needed.

Ions (K⁺, Na^\+, Cl^-, NO₃^-, PO₄^3-, etc.). The concentration of potassium in the vacuole can be tens of times higher than in the cytosol, which is important for osmoregulation and enzyme activity (Andreev, 2001; Maeshima, 2001).

Sugars (glucose, fructose, sucrose). In sugar beet and sugarcane, it is the vacuoles of the storage roots and stems that accumulate the main reserves of sucrose (Isayenkov, 2014; Neuhaus & Trentmann, 2014).

Organic acids (malic, citric, oxalic) — regulate pH and osmotic pressure.

Storage proteins (e.g., in aleurone grains of cereal and legume seeds). In seeds, these proteins are deposited in special protein vacuoles (or protein bodies), which are hydrolyzed during germination, providing the seedling with amino acids (Isayenkov, 2014; Evert, 2006).

Secondary metabolites (see section 1.4).

Degradation of macromolecules (proteins, nucleic acids, polysaccharides) that enter the vacuole from the cytoplasm.

Autophagy — digestion of damaged or obsolete organelles (mitochondria, peroxisomes, ER fragments). The organelle is surrounded by a membrane, fuses with the vacuole, and is broken down by its enzymes, while monomers are returned to the cytosol for reuse (Takatsuka et al., 2023; Aniento et al., 2022).

Programmed cell death (PCD). During aging or under stress, the vacuole may rupture, releasing hydrolases, which leads to cell autolysis (Andreev, 2001; Isayenkov, 2014).

Isolation of secondary metabolites — alkaloids, phenols, tannins, which are toxic to the cell itself at high concentrations. These substances accumulate in vacuoles and are used for defense against herbivores and pathogens (see section 1.6).

Sequestration of heavy metals (cadmium, lead, zinc). Special transporters move metal ions into the vacuole, where they bind to phytochelatins or organic acids (Khan et al., 2023; Isayenkov, 2014).

Regulation of cytosolic pH. The vacuole can take up or release protons (H^\+) through the action of proton pumps on the tonoplast, stabilizing cytoplasmic pH under acidification or alkalization (Andreev, 2001; Maeshima, 2001).

Regulation of Ca²⁺ ion concentration. In the cytosol, the calcium concentration must be very low (about 100 nM), while in the vacuole it can reach 10 mM. The vacuole acts as a calcium store, participating in signaling pathways: upon stimulation, Ca²⁺ is rapidly released from the vacuole through special channels (IP₃-gated), triggering the cell response (Andreev, 2001; Takatsuka et al., 2023).

Attract pollinators (insects, birds);

Participate in seed dispersal (bright fruits are eaten by animals);

Have antioxidant activity, protecting tissues from ultraviolet radiation and oxidative stress (Khan et al., 2023; Zhang et al., 2015).

Alkaloids (nicotine, caffeine, morphine) — nitrogen‑containing bitter poisons;

Tannins — impart an astringent taste and bind proteins in the oral cavity of insects;

Saponins and glycosides — disrupt digestion;

Calcium oxalate in the form of needle‑like crystals (raphides) mechanically damages mucous membranes when eaten (Khan et al., 2023; Mauseth, 2016).

Central (rounded or ellipsoid) vacuole — characteristic of most mature parenchyma cells. It pushes the cytoplasm toward the cell wall, forming a thin parietal layer.

Transvacuolar strands — thin cytoplasmic threads that pierce the central vacuole and connect the parietal layer of cytoplasm with the nucleus and other organelles (Strasburger et al., 1971; Serebryakova et al., 2006). They are stabilized by actin microfilaments and microtubules and can quickly disappear or appear when turgor changes.

Tubular vacuoles — observed in actively dividing cells (e.g., in tobacco BY‑2 cell culture) during mitosis. They form a temporary network around the spindle and later participate in cell plate formation (Takatsuka et al., 2023).

Fragmented (small) vacuoles — characteristic of cells undergoing plasmolysis (water loss), as well as some specialized tissues (e.g., endosperm cells at early developmental stages).

In young dividing cells, many small provacuoles are evenly distributed throughout the cytoplasm, and the nucleus is in the center.

As the cell grows and the central vacuole forms, the nucleus together with the nucleolus and most of the cytoplasm is pushed to the periphery and often becomes pressed against the cell wall (Mauseth, 2016; Graham et al., 2014).

In specialized cells (e.g., root hairs, moss cells, some parenchyma cells), the vacuole may be displaced toward one end, freeing space for apical growth (Takatsuka et al., 2023).

Fuse with other vacuoles (homotypic and heterotypic fusion);

Fragment into smaller compartments;

Form invaginations that can turn into internal vesicles (Takatsuka et al., 2023; Maeshima, 2001);

Change its shape depending on the cell cycle stage (see the section on BY‑2 culture).

Lysosomes — small membrane vesicles (0.2–0.5 μm) containing hydrolytic enzymes (acidic proteases, nucleases, lipases). They carry out intracellular digestion and autophagy but do not create osmotic pressure.

Endosomes — compartments involved in sorting of internalized membrane proteins and ligands.

Phagosomes and phagolysosomes — formed during phagocytosis of large particles (e.g., bacteria) and fuse with lysosomes.

Accumulating vacuoles are sometimes formed in specialized cells (e.g., in renal tubule cells during water diuresis, in osteoclasts during bone resorption), but they never occupy more than 10–20% of the cell volume and are not permanent structures (Graham et al., 2014).

Multiple vacuoles (from a few to several dozen) in one cell, especially in mycelial forms.

The tonoplast contains only V‑ATPase, but not V‑PPase (the latter is found only in some primitive fungi, but not in ascomycetes and basidiomycetes) (Maeshima, 2001).

Vacuoles are involved in regulation of the amino acid pool and ion homeostasis (especially Ca²⁺), as well as in detoxification (accumulation of dyes and toxins).

Fungal vacuoles can perform a storage function (glycogen, polyphosphates, arginine), but do not accumulate sucrose or anthocyanins.

Fungal vacuoles do not generate sufficient turgor pressure for growth by expansion; cell expansion occurs differently (apical growth involving the cytoskeleton and vesicles).

Contractile vacuoles — characteristic of freshwater protozoa (amoebae, euglenas, ciliates). They periodically fill with water entering from the cytoplasm along the osmotic gradient, then contract, expelling excess water to the outside. Thus, they perform an osmoregulatory function, preventing cell swelling and rupture (Strasburger et al., 1971; Graham et al., 2014). Contractile vacuoles are surrounded by a membrane but have a complex structure (a network of canals) and do not contain typical plant markers (TIP, V‑PPase). In some flagellates (e.g., Euglena), the contractile vacuole is located at the base of the flagellum.

Digestive vacuoles (phagosomes) — formed during endocytosis of food particles; they fuse with lysosomes and carry out intracellular digestion. Characteristic of heterotrophic protists (amoebae, ciliates, many flagellates).

Storage vacuoles — in some unicellular algae and euglenas, they can accumulate paramylon (a polysaccharide), chrysolaminarin, or fat. They are usually surrounded by a single membrane, but their transport systems are poorly studied.

SNARE proteins (e.g., VAMP, SYP) and their receptors, which ensure vesicle recognition and fusion (Isayenkov, 2014; Aniento et al., 2022).

RAB GTPases (ARA6, RAB₅, RAB7), which recruit tethering complexes (CORVET, HOPS) to membranes (Aniento et al., 2022).

Phosphoinositides (especially PI3P and PI(3,5)P₂), which create specific membrane domains that facilitate fusion (Zhang et al., 2015; Takatsuka et al., 2023).

Actin is necessary for maintaining the integrity of transvacuolar strands and for the directed movement of the nucleus in early G1 phase of the cell cycle. When actin is disrupted, the strands disappear and the nucleus loses its central position.

Microtubules participate in the formation of tubular vacuolar structures during mitosis, ensuring proper distribution of vacuolar material between daughter cells. They also direct the movement of vesicles carrying tonoplast proteins to the site of new cell plate formation (phragmoplast).

V‑ATPase uses the energy of ATP hydrolysis to pump H^\+ from the cytosol into the vacuole. This pump consists of many subunits and is sensitive to bafilomycin. It provides the main part of the gradient in vegetative cells.

V‑PPase uses the energy of inorganic pyrophosphate (PP_i) hydrolysis. It is a single polypeptide, absent in animals and fungi, but widespread in plants. V‑PPase is especially active in early development (seedlings, endosperm) and under ATP deficiency.

During programmed cell death (PCD) during differentiation of xylem conducting elements (vessels and tracheids), the tonoplast is first disrupted, and hydrolytic enzymes enter the cytoplasm, causing cell autolysis. Only the lignified cell wall remains, through which water moves (Andreev, 2001; Isayenkov, 2014).

During autophagy in leaf development (e.g., in dicotyledons during formation of interveinal spaces), vacuoles fuse and break down, creating space for aerenchyma.

Genetic regulation — through expression of genes for tonoplast proteins (V‑ATPase, TIP, SNARE, RAB). In mutants of genes VACUOLELESS1, VTI11, VPS41, central vacuole formation is disrupted, cells become very small and often die (Isayenkov, 2014; Aniento et al., 2022).

Signaling systems — auxin, brassinosteroids, and abscisic acid affect vacuolar fusion via ROP GTPases and phosphoinositide turnover (Takatsuka et al., 2023).

Osmotic status — during drought, decreased turgor activates signaling cascades (e.g., MAPK pathways), leading to actin reorganization and changes in vacuole shape (Neuhaus & Trentmann, 2014).

Supply of membrane material. Newly formed regions of the tonoplast are delivered as transport vesicles that bud off from the trans face of dictyosomes (Golgi) and, in some cases, directly from the ER (Isayenkov, 2014; Strasburger et al., 1971).

Delivery of soluble vacuolar proteins. Most hydrolytic enzymes and storage proteins are synthesized on the rough ER, then pass through the Golgi apparatus, where they receive carbohydrate tags (mannose‑6‑phosphate in animals, analogous signals in plants), are packaged into vesicles, and are directed to the vacuole. For this, there are specialized vacuolar sorting receptors (VSR) that recognize sorting signals on passenger molecules (Isayenkov, 2014).

Retrograde transport. Some proteins that mistakenly enter the vacuole can be returned to the Golgi or ER via COPI vesicles (Aniento et al., 2022). This ensures quality control of vacuolar contents.

Maintenance of transvacuolar strands. Thin cytoplasmic threads (transvacuolar strands) pass through the central vacuole, inside which bundles of actin microfilaments are located. Actin gives the strands rigidity and serves as tracks for the movement of vesicles and organelles (for example, the nucleus can migrate along these strands). When actin is disrupted (e.g., with latrunculin), the strands disappear and the nucleus loses its central position.

Formation of the vacuolar network during mitosis. During cell division, the spindle and phragmoplast microtubules participate in vacuole fragmentation and subsequent redistribution of vacuolar material between daughter cells. Artificial disruption of microtubules (with oryzalin) disturbs this process.

Anchoring of the vacuole to the plasma membrane. Cortical microtubules and actin filaments hold the vacuole in a specific position, especially in cells with apical growth (root hairs, pollen tubes).

Mechanical connection is achieved via transvacuolar strands, which connect the parietal cytoplasm with the nucleus. The nucleus is “suspended” in the central vacuole on these strands, allowing it to remain approximately in the center of the cell even when the vacuole is very large (Takatsuka et al., 2023; Mauseth, 2016). During plasmolysis, the nucleus together with the cytoplasm detaches from the wall but retains contact with the vacuole via the tonoplast.

Metabolic connection is determined by the fact that the nucleus controls the expression of genes for tonoplast proteins (V‑ATPase, TIP, transporters, SNARE) and for enzymes that synthesize secondary metabolites accumulated in the vacuole. In turn, signals from the vacuole (e.g., changes in osmotic pressure, increased concentration of Ca²⁺ or toxins) can modulate nuclear transcriptional activity via calcium and kinase cascades (Andreev, 2001; Neuhaus & Trentmann, 2014).

Metabolite exchange. Sugars and organic acids can enter the cytosol from the vacuole and then be taken up by mitochondria (respiration) and plastids (fatty acid and amino acid synthesis). Conversely, breakdown products (ammonia, CO₂) can be temporarily stored in the vacuole.

Calcium signaling. Mitochondria and chloroplasts, like the vacuole, participate in regulating cytosolic Ca²⁺. Upon a stimulus, Ca²⁺ can be released simultaneously from the vacuole and from these organelles, coordinately triggering the cellular response (Andreev, 2001).

Autophagy. Under starvation or aging, damaged mitochondria and plastids can be captured by autophagosomes and digested in the vacuole (Takatsuka et al., 2023). This process is especially active in senescing leaves and under stress.

Coordination of transport. The plasma membrane actively pumps ions (K⁺, Cl^-, NO₃^-) into the cytosol, and the vacuole then accumulates them, creating an osmotic gradient. This is especially evident in stomatal guard cells: during stomatal opening, K^\+ rapidly enters the cytosol through the plasma membrane and then into the vacuole through the tonoplast, causing water influx and an increase in guard cell volume (Takatsuka et al., 2023; Graham et al., 2014).

Vesicle fusion. Exocytosis (secretion of mucus, nectar, cell wall materials) begins with vesicles budded from the Golgi apparatus; they may first fuse with the vacuole, and then the vacuole fuses with the plasma membrane (common pathway). In addition, during cell expansion, the vacuolar and plasma membranes increase synchronously by inserting new portions of lipids and proteins from intracellular pools.

Response to osmotic stress. During dehydration (drought, salinity), the plasma membrane loses water and the tonoplast contracts, triggering plasmolysis. When water supply is restored, the vacuole rapidly swells, returning the protoplast to its original state. This reversibility is possible due to the high elasticity of the tonoplast and the presence of TIP aquaporins.

Ions and small molecules that have exited the vacuole into the cytosol can pass through plasmodesmata into neighboring cells.

Transvacuolar strands often extend to plasmodesmata, connecting the nucleus of one cell with the cytoplasm of its neighbor.

Accumulation of osmolytes. Under water deficit, osmotically active substances — proline, sugars (sucrose, fructose), glycine betaine, mannitol and others — accumulate in vacuoles (Serebryakova et al., 2006; Strasburger et al., 1971). These compounds do not disrupt metabolism (they are “compatible osmolytes”) and help the vacuole retain water even when the soil dries out. Breeding for an increased ability to synthesize and accumulate proline and sugars in vacuoles is one approach to creating drought‑tolerant varieties.

TIP aquaporins. The tonoplast contains a large number of water channels (TONOPLAST INTRINSIC PROTEINS, TIP). Regulation of their activity (e.g., by phosphorylation) allows the cell to quickly adapt to changes in water potential. Varieties with increased expression of certain TIP isoforms recover turgor better after drought (Maeshima, 2001; Neuhaus & Trentmann, 2014). In transgenic plants, enhanced expression of TIP often increases drought tolerance.

V‑ATPase and V‑PPase. Maintaining the gradient required for osmolyte accumulation requires active proton pumps. Genetic enhancement of V‑ATPase or V‑PPase subunit expression may promote better osmoregulation under drought (Neuhaus & Trentmann, 2014; Maeshima, 2001).

Na⁺/H^\+-antiporters (e.g., AtNHX1 in Arabidopsis) are localized on the tonoplast and transport Na⁺ from the cytosol into the vacuole in exchange for H^\+. Creating transgenic plants with increased expression of NHX1 (in rice, wheat, tomato, rapeseed) significantly increases their tolerance to salinity (Isayenkov, 2014). This approach is already used in biotechnology.

Enhancement of V‑ATPase and V‑PPase. Since the activity of the Na⁺/H^\+-antiporter depends on the transmembrane proton gradient, co‑expression of V‑ATPase (subunits A, B, C) or V‑PPase genes together with NHX1 leads to an additive effect — increased Na^\+ accumulation in the vacuole and greater salt tolerance (Maeshima, 2001; Neuhaus & Trentmann, 2014).

Breeding for increased antiporter activity. In naturally salt‑tolerant species (e.g., quinoa, sugar beet), the expression level of NHX and the activity of V‑ATPase are markedly higher than in sensitive crops. These traits can be used in marker‑assisted selection.

Sugar transporters on the tonoplast. The main carriers of glucose and fructose are proteins of the TMT (Tonoplast Monosaccharide Transporter) family. In Arabidopsis, three members of this family have been identified; a similar system is presumed in crop plants (Neuhaus & Trentmann, 2014; Wormit et al., 2006, cited in Neuhaus). Studying polymorphisms in TMT genes may assist in breeding for high sugar content.

Regulation of TMT activity. Phosphorylation of TMT by the protein kinase VIK1 (MAPKKK) stimulates glucose uptake by the vacuole (Wingenter et al., 2011, cited in Neuhaus). This opens opportunities for controlling fruit sweetness by modulating this regulatory cascade.

Sucrose accumulation. Sucrose is transported into the vacuole via other carriers (e.g., SUC4 in Arabidopsis, VSUC1 in sugar beet). In sugar beet, breeding for high sugar content has led to the selection of forms with increased activity of these carriers and enhanced expression of V‑ATPase (Endler et al., 2006, cited in Neuhaus).

Organic acids and fruit taste. In grapes, apples, tomatoes, acidity (malic/citric acid ratio) is determined by vacuolar transport. Malate channels and antiporters on the tonoplast regulate the efflux or accumulation of acids depending on the ripening stage. Understanding these processes allows breeding for varieties with optimal flavor.

Genetic engineering of the anthocyanin pathway. In tomato, potato, rice, maize, and other crops, transgenic lines with increased anthocyanin content in fruits or tubers have been obtained by introducing biosynthesis genes (e.g., MYB transcription factor genes, CHS, DFR, ANS). Anthocyanin accumulation occurs precisely in vacuoles, and retention requires a low pH and the presence of transporters (ABC transporters, MATE transporters) (Sunil & Nandini, 2022).

Effect of vacuolar pH on coloration. Anthocyanin color depends on pH: at pH < 3 they are red, at pH 5–6 they are purple or blue. Changes in V‑ATPase and V‑PPase activity in petal vacuoles (in hydrangea, petunia, geranium) shift the pH and change flower color. This is used in horticulture to produce “blue” forms (e.g., acidifying the soil for hydrangeas).

Detoxification mechanism. Heavy metal ions are bound in the cytosol to phytochelatins (cysteine‑rich peptides) or glutathione, and then the complex is actively transported into the vacuole by ABC transporters (e.g., AtABCC1, AtABCC2) (Andreev, 2001). In the vacuole, metals additionally bind to organic acids and tannins.

Hyperaccumulator plants. Species of the genus Thlaspi, Arabidopsis halleri, Sedum alfredii can accumulate in leaf vacuoles up to 3% Cd, 1% Ni, or 5% Zn on a dry weight basis without visible damage (Khan et al., 2023). Studying the genes of their transporters allows the transfer of these traits to crop plants or the use of the plants themselves for phytoextraction.

Practical applications. Growing such plants on contaminated landfills, around industrial zones, on fields after excessive fertilizer application (containing cadmium) allows metal extraction; the plant biomass is then disposed of (incineration with metal recovery). This method is cheaper and more environmentally friendly than chemical or mechanical cleaning.

Tannins, alkaloids, glycosides, calcium oxalate crystals (raphides) are stored in vacuoles and released upon cell damage, repelling or poisoning herbivores. Breeding for increased content of these compounds can improve variety resistance (but it should be noted that this may be undesirable for humans and livestock).

Glucosinolates (in crucifers) and their hydrolysis products (isothiocyanates) accumulate in vacuoles, while the enzyme myrosinase is located in cytoplasmic compartments. When tissue is chewed, cells break down, the enzyme contacts the substrate, and a toxic substance is formed that repels caterpillars and beetles. Genetic engineering can strengthen this defense mechanism.

ABC transporters, in addition to detoxifying xenobiotics, participate in diverting phytoalexins (antibiotic compounds synthesized upon pathogen infection) into the vacuole, thereby increasing resistance to fungal and bacterial diseases (Andreev, 2001).

Selenium. Selenium can replace sulfur in metabolism and accumulate in vacuoles as selenate, selenite, or selenoamino acids. Consumption of selenium‑rich plants (e.g., wheat, rice, broccoli) is beneficial for the immune system and for the prevention of some diseases. Breeding for increased selenium accumulation in grain and leaves is a promising direction.

Iodine. Although iodine is not essential for plants, some marine algae accumulate it in vacuoles in huge amounts (up to 1% dry mass). In terrestrial crops, iodine accumulation can be enhanced by applying iodide fertilizers and selecting genotypes with active iodine transporters on the tonoplast. This could help solve the problem of iodine deficiency in regions where the population does not get enough iodine from food.