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Cellular Inclusions

Last updated: May 30, 2026EspañolРусский

When examining a living plant cell under a microscope, two types of structures immediately catch the eye. The first are permanent, vital components: the nucleus, chloroplasts, mitochondria. The second are something entirely different: bright oil droplets, starch grains, glistening crystals, or colourless protein aggregates. These structures appear and disappear in the cell, are not strictly necessary for its immediate survival, but play a huge role in the life of the whole plant. These are cellular inclusions.

In scientific botanical literature, the term ergastic substances (from Greek ergon – work, product of activity) is often used to describe them (Yakovlev et al., 2006). Cellular inclusions are products of protoplast metabolism that accumulate in the cell as grains, droplets, crystals, or amorphous masses. The main difference between inclusions and organelles is that they are generally not “living” structures. They lack their own membranes (or possess them only transiently), have no DNA of their own, and cannot reproduce independently (Evert, 2006).

A simple analogy can be drawn. If we imagine the cell as a high-tech factory, the organelles are its workshops and laboratories (machines, reactors, an office with blueprints). Cellular inclusions are the finished products (bags of grain, tanks of oil, packaged goods in a warehouse) or, in some cases, containers of waste awaiting disposal.

By their nature, inclusions are divided into three main groups:

  1. Reserve (trophic) substances: starch, proteins, oils. This is the cell’s “strategic reserve”.

  2. Defensive substances and secondary metabolites: essential oils, resins, alkaloids, tannins, which make the plant bitter, poisonous, or resistant to rot.

  3. Final products of metabolism (excretes): crystals of calcium oxalate, calcium carbonate, silica, which the cell “entombs” in its vacuole, detoxifying harmful excesses of ions.

Below we will examine in detail each of the main types of cellular inclusions, their structure, and their role in the life of crop plants.

1. Evolutionary origin: from sea to land

Why did plants begin to accumulate these substances in such significant quantities? The answer lies in the evolutionary transition from an aquatic to a terrestrial lifestyle.

For ancient algae living in a stable aquatic environment, resource scarcity was not such an acute problem. However, the move onto land about 450 million years ago presented the first plants (psilophytes and their descendants) with harsh conditions:

  1. Irregular nutrition. Soil is poor, water is alternately present and absent. The need arose to store nutrients in advance for periods of drought or cold. Thus, storage forms of carbohydrates (starch) and lipids (oils) evolved.

  2. The need for protection. Moving onto land meant the appearance of herbivorous animals and phytopathogenic fungi and bacteria. By accumulating poisonous alkaloids, bitter tannins, or sharp calcium oxalate crystals in their vacuoles, plants developed a powerful mechanism to deter enemies (Khan et al., 2023). For example, oxalate crystals are one of the most ancient forms of defence, found even in primitive ferns.

  3. Management of ion homeostasis. On land, plants began to absorb excess calcium and other minerals from the soil. There was nowhere to simply “throw them away”. The solution was to bind toxic Ca2+ ions with oxalic acid synthesised in the cell, forming insoluble and inert calcium oxalate. Crystals of this salt became the very “graveyard” where the plant isolates hazardous waste (Graham et al., 2014).

The key evolutionary acquisition for realising these functions was the central vacuole. It is here, separated from the active cytoplasm by the tonoplast membrane, that plants accumulate most of their ergastic substances. Moreover, modern research shows that the vacuole is not just a passive storehouse but a dynamic hub that regulates the accumulation of inclusions in response to environmental signals (Aniento et al., 2022). It can change its pH and activate enzymes that break down storage proteins when a seed germinates.

Thus, the ability to accumulate diverse inclusions became an evolutionary advantage, allowing plants to colonise land and adapt to an endless variety of climatic conditions and biological threats.

The study of cellular inclusions has enormous applied significance for agronomy. It is thanks to them that we assess crop quality: the gluten content in wheat grain (storage protein) or the starch content in potato tubers determines their nutritional value. Understanding how and why a plant synthesises particular substances enables breeders to develop varieties with improved properties and high resistance.

Below we will examine in detail each of the main types of cellular inclusions, their structure, and their role in the life of crop plants.

2. Classification of cellular inclusions

As we have already established, cellular inclusions (ergastic substances) are extremely diverse in their nature. To systematise this knowledge, botany employs two main approaches to classification: by chemical composition (what is the substance?) and by functional role (what role does the substance play in the life of the plant?). Both systems complement each other.

2.1. Classification by chemical nature

This approach is the primary one for agricultural sciences, because it is the chemical composition of inclusions that determines their nutritional, fodder, or technical value. According to this principle, all ergastic substances are divided into several major groups (Yakovlev et al., 2006).

1. Carbohydrates

  • Starch: the main storage polysaccharide of higher plants. Deposited as characteristic grains in amyloplasts (a special type of leucoplast). Starch forms the basis of the harvest of cereal crops and potatoes.

  • Inulin: a storage polysaccharide typical of plants of the Asteraceae family (Asteraceae), for example, chicory and Jerusalem artichoke. Unlike starch, inulin is soluble in hot water and is deposited not in plastids but in soluble form in vacuoles.

  • Sugars (sucrose, glucose, fructose): often accumulate in the cell sap of vacuoles, providing the sweet taste of fruits and storage organs (beet, sugarcane).

2. Lipids (fats and oils)

  • Represent the most energy-dense form of storage. Deposited in the cytoplasm as small droplets – spherosomes or oleosomes. Seeds of oilseed crops (sunflower, rapeseed, flax, soybean) are particularly rich in oils. From a biochemical perspective, oils are esters of glycerol and unsaturated fatty acids (Maeshima, 2001).

3. Proteins (storage proteins)

  • Protein accumulation in seeds is a critically important process for germination and human nutrition. Storage proteins are most often deposited as aleurone grains in the vacuoles of seeds (Evert, 2006). In legumes and cereals, it is these proteins that determine the nutritional value of grain and flour (gluten).

4. Mineral crystals

  • Calcium oxalate (CaC2O4): the most common form. Occurs as single crystals (styloids, prismatic crystals), druses (aggregates), raphides (bundles of needle-like crystals), or crystal sand.

  • Calcium carbonate (CaCO3): occurs less frequently, often as cystoliths – grape-like outgrowths on the cell wall.

  • Silica (SiO2): accumulates in the cell walls of cereals (rice, wheat) and horsetails, giving them strength and rigidity (Khan et al., 2023).

5. Secondary metabolites

  • This is a huge group of substances synthesised by the plant but not directly involved in primary metabolism (growth, respiration). They include alkaloids (caffeine, nicotine, morphine), tannins, essential oils, resins, and phenolic compounds, including anthocyanins (plant pigments) (Neuhaus & Trentmann, 2014). Most of them accumulate in vacuoles.

2.2. Classification by functional role

From the perspective of plant physiology, it is more convenient to divide inclusions according to their role in metabolism and adaptation.

  • Reserve (trophic) substances: perform the function of an energy and plastic reserve. Used by the plant during dormancy, seed germination, fruit formation, and active growth phases. Examples: starch, oils, storage proteins.

  • Defensive substances: serve to deter herbivores (alkaloids, tannins), suppress the growth of competing plants (allelopathy), or protect against pathogens (phytoalexins). This also includes mechanical defensive structures, such as calcium oxalate raphides, which, when they enter an insect’s mouth, cause mechanical irritation (Khan et al., 2023).

  • Secretory products (excretes): substances that are either discharged from the cell (nectar, essential oils) or isolated inside the cell as end products of metabolism (calcium oxalate crystals). Their accumulation is a means of detoxification and maintaining ion balance.

  • Pigments: a special functional group. Although chlorophyll is a plastid pigment, many bright pigments (anthocyanins) are cellular inclusions. They accumulate in vacuoles and serve to attract pollinators and seed dispersers, as well as to protect tissues from ultraviolet radiation.

2.3. Classification by localisation within the cell

For the diagnosis of plant material, it is important to know where exactly in the cell a given inclusion is located.

  • In plastids (amyloplasts): starch.

  • In the cytoplasm (spherosomes): fats and oils.

  • In vacuoles: proteins (aleurone grains), sugars, organic acids, anthocyanins, alkaloids, tannins, as well as calcium oxalate crystals and cystoliths.

  • In the cell wall: silica (phytoliths).

In the following sections, we will focus in detail on each of these types to understand how their structure relates to their functions and what significance they have for agricultural practice.

3. Overview of the main types of cellular inclusions

In this section, we will sequentially examine the most important groups of cellular inclusions found in plants. For each type, we will discuss its chemical nature, morphology (form and structure), localisation within the cell, biological function and, particularly important for agronomy, its significance for humans and agricultural production.

3.1. Carbohydrate inclusions

Carbohydrates are the primary products of photosynthesis and the main energy source for the cell. Some carbohydrates are used immediately by the plant, while others are stored “for later” as characteristic structural formations. The main forms of storage carbohydrates in the plant kingdom are starch and, to a lesser extent, inulin.

Starch

Starch granules in Arabidopsis leaf cells
CC BY 4.0

Starch granules in <span lang="la" class="biological-name">Arabidopsis thaliana</span> leaf cells

International Journal of Molecular Sciences (MDPI)[https://www.mdpi.com/1422-0067/22/11/5666]

Starch is the principal reserve polysaccharide of the vast majority of higher plants. It is a polymer of α-D-glucose and occurs as characteristic grains (granules), the shape and size of which are often species‑specific. Due to this property, starch grains are used in the microscopic diagnosis of plant material (Yakovlev et al., 2006).

Origin and localisation: two starch pools. In a plant cell, starch can accumulate in two different types of plastids, which determines its function:

  1. Assimilation (primary) starch: synthesised in the chloroplasts of green leaves during photosynthesis (Graham et al., 2014). It is formed during daylight hours from excess glucose. Assimilation starch grains are very small, irregular in shape, and lack clear layering. Function: temporary deposition of carbohydrates, which are hydrolysed to sucrose at night and exported to other parts of the plant (roots, stems, fruits). This process provides for the growth and development of organs incapable of photosynthesis.

  2. Reserve (secondary) starch: synthesised in specialised leucoplasts – amyloplasts, which occur in storage tissues: the endosperm of cereals, potato tubers, root crops, and seeds of legumes. Reserve starch grains are typically large, with a layered structure resulting from the daily rhythm of synthesis (alternating layers of different densities) (Evert, 2006).

Structure of the starch grain. The starch grain has concentric layers around a centre – the hilum (the point from which grain formation began). Three types of grains are distinguished:

  • Simple grains: have a single crystallisation centre (one hilum). Characteristic of potato, wheat, rye.

  • Compound grains: consist of several simple grains “glued” together, each with its own hilum. Typical of oats, rice, buckwheat.

  • Semi‑compound grains: initially form as simple, then a common envelope begins to be deposited around them. Found, for example, in maize.

In polarised light, starch grains exhibit birefringence, visible as a characteristic “Maltese cross”. This demonstrates the presence of crystalline zones (amylopectin) and amorphous zones (amylose) within them (Graham et al., 2014).

Starch dynamics: formation and utilisation. Starch is formed in amyloplasts by the action of the enzyme starch synthase, which elongates glucose chains. Utilisation (mobilisation) of starch begins when the plant requires energy or carbon skeletons for growth. During seed germination, for example, amylases (enzymes that hydrolyse starch) break down the storage starch of the endosperm to maltose and then to glucose, which is transported to the growing embryo (Maeshima, 2001).

In leaves, the dynamics of assimilation starch follow a diurnal pattern: it is synthesised during the day and hydrolysed at night. Disruption of this balance can lead to excessive starch accumulation (“starchiness”) and reduced yield.

Agronomic significance. Starch is a key component of the harvest for most agricultural crops. It is the starch content that determines:

  • Yield of cereals (wheat, rice, maize, barley).

  • Quality of potato tubers (the higher the starch content, the higher the nutritional value and the better the processing properties for starch or chips).

  • **Nutritional value of legume seeds and root crops.

From the perspective of plant physiology, understanding the processes of starch synthesis and degradation allows us to manage the accumulation of dry matter in the marketable part of the crop, which is directly linked to the productivity of agrocenoses.

Inulin

Inulin is a reserve polysaccharide built from β-D-fructose residues. Unlike starch, it is readily soluble in hot water and is deposited not in plastids but in dissolved form in the vacuoles of storage tissues (Stern & Jansky, 2021).

Distribution and functions. Inulin is typical of plants of the Asteraceae family (Asteraceae), as well as some members of the Campanulaceae and Lobeliaceae. The greatest agronomic importance is found in:

  • Jerusalem artichoke (Helianthus tuberosus) – its tubers are rich in inulin.

  • Chicory (Cichorium intybus) – roots contain up to 60% inulin on a dry weight basis.

  • Dandelion (Taraxacum officinale).

The function of inulin is analogous to that of starch – it is a storage carbohydrate. However, its solubility and chemical structure determine its physiological role in cell osmoregulation, helping the plant withstand drought and salinity (Graham et al., 2014).

Applied significance. Inulin is of interest to the food industry and medicine as a prebiotic (stimulates the growth of beneficial gut microflora) and as a sugar substitute for diabetics (since it does not cause a sharp rise in blood glucose levels). In addition, hydrolysis of inulin yields fructose, which is sweeter than sucrose and is used as a low‑calorie sweetener. Processing Jerusalem artichoke and chicory for inulin is a promising branch of agricultural biotechnology.

In the next section, we will examine another important group of inclusions – lipid inclusions (fats and oils), which play a key role in the energy balance of plants and humans.

3.2. Lipid inclusions (fats and oils)

Lipid inclusions are the second most important (after starch) form of energy storage in the plant world, and in many species (for example, in oilseeds) they are the most important. Unlike carbohydrates, lipids are high‑energy compounds that release approximately twice as much energy per unit mass when oxidised. Therefore, plants, and especially their seeds, often store lipids to provide energy to the young seedling until it transitions to independent photosynthesis.

Chemical nature and forms of inclusions

The basis of plant oils and fats is composed of esters of the trihydric alcohol glycerol and higher fatty acids – triacylglycerols (Maeshima, 2001). The difference between oils (liquid at room temperature) and fats (solid) is determined by the ratio of saturated to unsaturated fatty acids in their composition: a predominance of unsaturated (oleic, linoleic, linolenic) acids makes the oil liquid, while a predominance of saturated (palmitic, stearic) acids makes it solid.

In the cell, lipid inclusions appear as small, strongly light‑refracting droplets. Under electron microscopy, they appear as electron‑transparent spheres, lacking their own membrane but surrounded by a monolayer of phospholipids and special proteins – oleosins (Evert, 2006). Such droplets are called oleosomes or spherosomes. They can be scattered throughout the cytoplasm but are more often concentrated in specific zones of storage tissue.

Formation and localisation

Fatty acid biosynthesis occurs in plastids (primarily leucoplasts and chloroplasts). The fatty acids are then transported to the endoplasmic reticulum (ER), where they are assembled into triacylglycerol molecules (Graham et al., 2014). The oleosomes themselves also form in the ER: oil droplets “bud off” from the membranes of the smooth ER and emerge into the cytoplasm, covered by a monolayer of lipids and oleosin proteins.

Localisation of lipid inclusions varies greatly depending on the tissue type:

  • In seeds of oilseed crops (sunflower, rapeseed, flax, soybean, peanut), oleosomes occupy most of the volume of endosperm or cotyledon cells. They often neighbour aleurone grains (storage proteins) and starch grains.

  • In fruits (e.g., avocado, olive), lipids also accumulate in the flesh.

  • In vegetative organs (roots, stems, bulbs), lipid inclusions are less common and in smaller quantities, but in some succulents and perennials they may serve as a reserve for the winter period.

Functions of lipid inclusions

  1. Energy reserve: oxidation of 1 g of fat releases about 39 kJ of energy (compared to 17 kJ from oxidation of 1 g of starch). This makes lipids the most “economical” form of energy storage under the limited space of a seed.

  2. Source of water: oxidation of fats (via lipolysis and β‑oxidation) produces a significant amount of metabolic water. For seedlings growing in dry soil, this can be critically important (Graham et al., 2014).

  3. Structural function: lipids are components of membranes, but in this context, they serve as a reserve of precursors for membrane lipid synthesis during rapid growth.

  4. Defensive function: in some cases, lipid droplets may incorporate lipophilic compounds that are toxic to insects and fungi.

Utilisation (mobilisation) of lipids

During germination of oil‑rich seeds (e.g., sunflower or castor bean), storage triacylglycerols are hydrolysed by the enzyme lipase to glycerol and free fatty acids. The fatty acids then enter specialised organelles – glyoxysomes (a type of peroxisome), where they are broken down via the glyoxylate cycle to produce succinate. Succinate is converted into carbohydrates (glucose), which are used by the seedling until green leaves appear. Thus, in oilseed plants, lipids serve as the raw material for gluconeogenesis – the synthesis of carbohydrates from non‑carbohydrate precursors (Evert, 2006).

Interconnection with other cell components

Lipid inclusions are closely linked to the endoplasmic reticulum (site of biosynthesis) and to mitochondria and glyoxysomes (sites of oxidation during germination). The dynamics of lipid inclusions are also hormonally regulated: abscisic acid (ABA) induces oil accumulation in developing seeds, while gibberellins activate their hydrolysis during germination (Maeshima, 2001).

Agronomic and economic significance

It is precisely due to their ability to accumulate lipids that many crops are called oilseeds. They are the raw material for the production of vegetable oils, used for food, technical purposes (biodiesel, lubricants, drying oils), and in medicine.

  • Major oilseed crops: sunflower (Helianthus annuus), rapeseed (Brassica napus), soybean (Glycine max), flax (Linum usitatissimum), peanut (Arachis hypogaea), olive (Olea europaea).

  • Breeding aspects: for the agronomist, not only the total oil content but also its fatty acid composition (the ratio of oleic, linoleic and linolenic acids) is important. For example, a high content of erucic acid in rapeseed oil is unacceptable for food purposes, so varieties (canola) with low erucic acid content have been bred.

  • Crop quality: growing conditions (temperature, irrigation, mineral nutrition) strongly influence oil accumulation. For instance, nitrogen fertilisation increases yield but may reduce oil content; heat and water stress during seed filling, on the contrary, often stimulate fat accumulation.

Understanding the physiology of lipid accumulation and mobilisation helps the agronomist to plan the cultivation technology of oilseed crops appropriately in order to obtain the maximum yield of high‑quality oil.

Next, we will consider the next major group – protein inclusions (aleurone grains), which are of paramount importance for grain quality and feedstuffs.

3.3. Protein inclusions (aleurone grains)

Protein inclusions are a form of storage protein accumulation in plant cells. Unlike starch and oils, proteins serve not so much as an energy reserve but as a plastic (structural) reserve, providing young growing tissues with amino acids – the building blocks for the synthesis of new enzymes, structural and regulatory proteins. Protein inclusions are particularly important for seeds – they determine the nutritional value of cereals and legumes.

Chemical nature and types of storage proteins

Plant storage proteins belong to the group of simple proteins (proteins). By solubility, they are divided into several fractions, which has diagnostic and technological significance (Yakovlev et al., 2006):

  • Albumins – soluble in water. Found in many seeds, but are rarely the main storage form.

  • Globulins – soluble in salt solutions. These are the main storage proteins of legumes (e.g., legumin and vicilin in pea and soybean) and many other plants. Globulins are often deposited as crystalloids.

  • Prolamins – soluble in alcohol. Characteristic of cereals: gliadin (wheat), zein (maize), hordein (barley). It is the prolamins (in complex with glutenins) that form gluten, which determines the baking properties of flour.

  • Glutelins – soluble in alkalis or acids. Also part of the gluten of wheat and other cereals.

Localisation and structure: aleurone grains

Storage proteins accumulate in the vacuoles of seeds and some vegetative organs. During seed maturation, the vacuoles dehydrate, and the proteins they contain precipitate, forming dense structures – aleurone grains (Evert, 2006). An aleurone grain is essentially a dried, dehydrated protein vacuole.

Based on their internal structure, aleurone grains are divided into three types:

  1. Simple – consist only of an amorphous protein matrix. Such grains are typical, for example, of legume seeds.

  2. Compound – in addition to an amorphous matrix, contain one or more crystalloids (crystalloid‑like protein bodies) and one or more globoids (spherical inclusions).

    • Crystalloids are aggregates of the storage proteins themselves (usually globulins), having a regular but not always truly crystalline structure (they are capable of swelling in water).

    • Globoids contain phytin (a salt of inositol hexaphosphoric acid with calcium and magnesium). This is the main depot of phosphorus, potassium, magnesium and calcium in the seed (Graham et al., 2014). Globoids are usually spherical or irregular in shape and contain no protein.

  3. Mixed type – combine both variants.

Crystalloids and globoids can be arranged inside the aleurone grain in various combinations, which allows their structure to be used for seed diagnostics (Isayenkov, 2014).

Formation and mobilisation

Storage proteins are synthesised on the rough endoplasmic reticulum (RER) as large precursors (proproteins). They are then transported via the Golgi apparatus (or sometimes bypassing it) to vacuoles, where they mature (proteolytic removal of signal peptides). In the mature seed, aleurone grains are in a “resting” state.

Upon seed germination, hydrolytic enzymes (proteases and peptidases) are activated. The crystalloids and protein matrix are broken down into free amino acids. Phytin, under the action of phytase, is hydrolysed, releasing phosphate, cations and inositol. All these products are transported to the growing embryo, providing it with nitrogen, sulfur, phosphorus and metals (Evert, 2006). This process is strictly regulated by phytohormones: gibberellins stimulate hydrolysis, abscisic acid inhibits it.

Interconnection with other cell components

In cereal endosperm cells, a close spatial and functional relationship is often observed between aleurone grains (storage protein) and amyloplasts (storage starch), as well as oil droplets. In some seeds, a single aleurone grain may contain both a protein crystalloid and a phytin globoid, and even small starch grains (Yakovlev et al., 2006). This is a unified “storehouse” of nutrients.

Agronomic and economic significance

Understanding the nature of protein inclusions is key to managing the quality of agricultural products.

  1. Wheat grain quality: the content and properties of prolamins (gliadins) and glutelins (glutenins) determine the flour strength (ability to form elastic dough). Breeding for high gluten content is a priority for the baking industry. Conversely, for feed and food purposes (e.g., for the starch industry), varieties with reduced gluten are required.

  2. Nutritional value of legumes: soybean, pea, bean are rich in globulins. However, they often contain protease inhibitors (antinutritional factors) that block digestion. Heat treatment destroys these inhibitors, increasing protein digestibility.

  3. Phosphorus metabolism: phytin, contained in globoids, is the main form of phosphorus in seeds. However, phytin is poorly digestible by monogastric animals (pigs, poultry, humans) and can pollute the environment with phosphates. Therefore, breeding for low‑phytate varieties (or using the enzyme phytase in feed) is an important task in agronomy and biotechnology.

  4. Industrial use: besides milling, storage proteins are used to produce plant isolates (soy protein), in food additives, and for the production of bioplastics and adhesives.

In the following sections, we will consider mineral crystalline inclusions (calcium oxalate, silica) and secondary metabolites (alkaloids, essential oils, tannins), which play a key role in plant defence.

3.4. Crystalline inclusions

Crystalline inclusions are one of the most enigmatic and, at first glance, “useless” groups of ergastic substances for the plant. However, this is not the case. They perform crucial functions: from the elimination of toxic ions to protection against herbivory and even tissue strengthening. Unlike organic inclusions (starch, oil, protein), crystals are inorganic salts or oxides that are deposited primarily in vacuoles, and sometimes in cell walls.

The main types of crystalline inclusions in plants are calcium oxalate (most common), calcium carbonate (less frequent), and silica (amorphous silicon dioxide).

Calcium oxalate (CaC2O4)

This is the most widespread form of mineral deposits in the plant kingdom. Calcium oxalate is practically insoluble in water, so once formed, it remains in the cell forever or is mobilised extremely slowly under severe calcium deficiency. Its crystals have diverse shapes, which are often a diagnostic feature for identifying a species or even a family of plants (Khan et al., 2023).

Forms of calcium oxalate crystals. Depending on the crystallisation conditions and the cell type (crystalline idioblasts), calcium oxalate can take the following forms (Evert, 2006; Yakovlev et al., 2006):

  • Raphides: bundles of very thin, needle‑like crystals, often pointed at both ends. Raphides are characteristic of many monocots (e.g., Liliaceae, Orchidaceae, Araceae) and some dicots. They are always surrounded by a mucous sheath and enclosed in a special crystal chamber inside the vacuole.

  • Druses: spherical aggregates consisting of many individual crystals (prisms) radiating from the centre, like sunbeams. Druses are widespread in dicots (e.g., in leaves of Ficus, in the bark and leaves of linden). They can reach considerable size and are clearly visible under light microscopy.

  • Prismatic crystals (styloids): single large crystals shaped like straight or oblique prisms, often with pointed ends. Found, for example, in onion bulbs, in roots and wood of many trees.

  • Crystal sand: a multitude of very small, angular or rounded crystals filling the entire vacuole. Characteristic of Solanaceae (e.g., deadly nightshade) and some Chenopodiaceae.

Formation and biological role. Calcium oxalate crystals form in specialised cells – crystalline idioblasts. The process begins with the plant accumulating calcium ions (Ca2+) in the vacuole. Oxalic acid, synthesised in the cytoplasm (mainly from ascorbic acid or via the glyoxylate pathway), also enters the vacuole. When a certain concentration is reached, Ca2+ and C2O42- ions combine to form the insoluble salt CaC2O4, which crystallises (Khan et al., 2023).

Main functions of oxalate crystals:

  1. Regulation of calcium homeostasis: plants absorb calcium passively from the soil with the water flow. Excess Ca2+ is toxic to the cytoplasm because it can disrupt enzyme function and signalling systems. Crystallisation is an effective way to “bind” and isolate excess calcium. However, the plant then loses the ability to mobilise it quickly, so this mechanism is often considered a form of excretion (elimination) via senescent or dying organs (leaves, bark) (Khan et al., 2023).

  2. Protection against herbivores: raphides and sharp prismatic crystals act as a mechanical barrier. When they enter the mouth of a caterpillar or the mucous membrane of an animal, they cause micro‑injuries, irritation and swelling. Raphides are often combined with toxic proteins (proteases) or alkaloids, enhancing the damaging effect. This is well known for members of the Araceae (e.g., Dieffenbachia) and Urticaceae (the stinging hairs of nettle contain oxalate crystals) (Khan et al., 2023).

  3. Detoxification of heavy metals: some studies have shown that calcium oxalate crystals can incorporate ions of toxic metals (cadmium, strontium, lead) into their crystal lattice, thereby reducing their damaging effect on the protoplast (Khan et al., 2023).

  4. Tissue strengthening: in old leaves, bark, and seed coats, crystals, especially druses and prisms, may play a reinforcing role, increasing the mechanical strength of tissues.

Calcium carbonate (CaCO3) and cystoliths

Calcium carbonate is less common than oxalate. It is also practically insoluble in water, but its crystals have a different shape.

The best‑known form of calcium carbonate in plants is the cystolith. These are not so much intracellular inclusions as outgrowths of the cell wall encrusted with calcium carbonate. Cystoliths form in specialised cells – lithocysts, which are usually larger than the surrounding cells and contain a large vacuole. The cell wall of the lithocyst invaginates into the cell, and on this outgrowth (the stalk) layers of calcium carbonate are deposited, forming a grape‑like or rounded body – the cystolith (Evert, 2006).

Cystoliths are characteristic of the families:

  • Urticaceae (Urticaceae) – Ficus, fig.

  • Moraceae (Moraceae).

  • Acanthaceae (Acanthaceae).

The functional significance of cystoliths is not fully understood. It is assumed that, like oxalate crystals, they serve to isolate excess calcium and possibly to strengthen the leaf (protection against damage).

Silica (SiO2) – phytoliths

Accumulation of amorphous silica in cell walls is a feature of many plants, especially cereals (Poaceae), sedges (Cyperaceae) and horsetails (Equisetaceae). Silica deposits are called phytoliths (from Greek phyton – plant and lithos – stone). They can take the form of small grains, rods, star‑shaped or complex sculpted structures (Mauseth, 2013).

Silica gives cell walls hardness and brittleness, protecting plants from herbivory (teeth are worn down, insect jaws are damaged). In addition, silicon deposits reduce water loss through transpiration and increase stem resistance to lodging. In agronomy, it is known that silicon fertilisers (silicates) increase the resistance of rice and oats to lodging and diseases.

The shape of phytoliths is often species‑specific, which is used in archaeology and palaeobotany to identify ancient grasses from silica remains in soil or animal dental calculus.

Formation and utilisation

Unlike calcium oxalate, crystalline inclusions were previously considered completely irreversible. However, modern research has shown that under severe deficiency of calcium or silicon, as well as during germination (to supply the embryo), some plants can partially dissolve crystals, mobilising the elements they contain (Khan et al., 2023). However, this ability is limited and not universal.

Agronomic significance of crystalline inclusions

  1. Microdiagnostics: the shape of calcium oxalate crystals and phytoliths is a reliable feature for distinguishing plant species, even in the absence of flowers or fruits. This is important for verifying the authenticity of medicinal raw materials and for seed testing.

  2. Pest resistance: breeding varieties with an enhanced ability to accumulate calcium oxalate crystals (especially raphides) or silica in leaves is one approach to creating insect‑ and mite‑resistant forms (Khan et al., 2023). However, it should be noted that crystals may reduce the forage value for livestock.

  3. Feed quality and food safety: high phytolith content in forage grasses (e.g., hay) reduces their digestibility and can cause dental problems in animals (especially horses). In some vegetables (e.g., rhubarb, spinach), high concentrations of calcium oxalate make them undesirable for people suffering from urolithiasis (oxalate kidney stones).

  4. Soil science: phytoliths, entering the soil after plant death, slowly decompose, returning silicon to the soil solution. This is part of the biogeochemical cycle of silicon. Knowledge of phytolith composition helps diagnose past vegetation types (palaeopedology).

In the next section, we will consider the most diverse and biologically active group of inclusions – secondary metabolites (alkaloids, tannins, essential oils and pigments), which play a key role in plant stress resistance.

3.5. Secondary metabolites as inclusions

In addition to primary metabolites (carbohydrates, lipids, proteins), plants synthesise a huge variety of low‑molecular‑weight organic compounds that are not directly involved in growth, respiration and reproduction. These are secondary metabolites (also sometimes called ergastic defensive substances). Unlike universal primary metabolites, secondary compounds are often species‑specific and perform specialised ecological functions.

Most secondary metabolites accumulate in vacuoles (if water‑soluble) or in specialised secretory structures – schizogenous (formed by cell separation) and lysigenous (formed by cell dissolution) cavities, resin ducts, essential oil cells and glandular hairs (Serebryakova et al., 2006; Evert, 2006).

Main groups of secondary metabolites

Alkaloids

This is a large group of nitrogen‑containing organic bases, often with a bitter taste and high physiological activity. More than 20,000 alkaloids are known (Serebryakova et al., 2006). They accumulate in vacuoles as salts of organic acids.

Functions:

  • Protection against herbivores: alkaloids are toxic or deterrent to insects, birds, mammals. For example, nicotine from tobacco is a neurotoxin, caffeine is an insecticide.

  • Allelopathy: suppression of competitor growth.

  • Growth regulation (at low concentrations).

Examples:

  • Nicotine (Nicotiana tabacum)

  • Caffeine (Coffea arabica, tea)

  • Morphine, codeine (Papaver somniferum)

  • Atropine (Atropa belladonna)

  • Scopolamine (Scopolia carniolica)

Agronomic significance: alkaloids are valuable medicinal substances, but they can also be undesirable in forage grasses (e.g., lupine alkaloids make its green matter bitter and toxic to livestock, although breeding has produced sweet varieties with low alkaloid content).

Phenolic compounds

This is the most numerous group of secondary metabolites, including phenolic acids, flavonoids, tannins and lignin (although lignin is not an inclusion but a cell wall component). Phenols are water‑soluble and accumulate in vacuoles.

Tannins – polymeric phenols with an astringent taste and the ability to precipitate proteins (turning hide into leather – tanning). Tannins protect plants from herbivory by insects and from fungal infections (Evert, 2006).

  • Localisation: in vacuoles of bark cells, leaves, unripe fruits, in specialised tannin idioblasts.

  • Examples: oak (bark), willow (bark), tea (leaves), bird cherry, unripe persimmon fruits.

Anthocyanins – water‑soluble flavonoid pigments that give colour to flowers, fruits, leaves (red, blue, purple, pink shades). Anthocyanins are glycosides: their molecule consists of an aglycone (anthocyanidin) and one or more sugar residues (Sunil et al., 2022).

  • Functions:

  • Attraction of pollinators and fruit dispersers.

  • Protection against ultraviolet (UV‑B) radiation.

  • Antioxidant defence under stress (cold, drought, pathogen attack).

  • Localisation: exclusively in vacuoles of epidermal cells and fruit flesh. The pH of the vacuole affects the colour of anthocyanins: in acidic medium – red, in neutral – violet, in alkaline – blue.

  • Examples: blueberry, red cabbage, rose petals, cornflower.

Terpenes (isoprenoids)

This is the largest class of natural compounds, built from isoprene units (C₅H₈). Terpenes include essential oils, resins, rubber, carotenoids (although carotenoids are chromoplast pigments, often classified as plastid pigments rather than vacuolar inclusions). Lipophilic terpenes accumulate not in vacuoles but in schizogenous or lysigenous cavities, resin ducts, essential oil cells (Strasburger, 1971; Serebryakova et al., 2006).

Essential oils – volatile mixtures of mono‑ and sesquiterpenes that give plants their characteristic odour.

  • Functions: attraction of pollinators, deterrence of herbivores (including large herbivores), antiseptic action (suppression of bacteria and fungi).

  • Localisation: in glandular hairs (lavender, mint, rosemary), in essential oil cells (citrus), in schizogenous cavities (St. John’s wort), in glandular spots (petunia).

  • Agronomic significance: essential oil crops are sources of essential oils for perfumery, medicine, and the food industry. Varieties with higher essential oil content are more resistant to pests.

Resins – viscous, often sticky mixtures of terpenoids, insoluble in water, hardening in air. They accumulate in resin ducts (canals), particularly well developed in conifers (pine, spruce, larch).

  • Functions: sealing wounds (tapping), protection against insects (bark beetles) and pathogens.

  • Examples: turpentine (terpentine + rosin), amber (fossilised resin).

Localisation and interconnections with other cell components

The vast majority of water‑soluble secondary metabolites (alkaloids, glycosides, phenols, anthocyanins) accumulate in vacuoles. The tonoplast contains specialised transport proteins (ABC transporters, antiporters) that actively pump these substances inside, maintaining their concentration many orders of magnitude higher than in the cytoplasm (Andreev, 2001). This allows:

  • Isolation of potentially toxic compounds from cytoplasmic enzymes.

  • Creation of a depot of biologically active substances that are released only when the cell is damaged (e.g., when the vacuole is ruptured by an insect).

Lipophilic terpenes (essential oils, resins) accumulate outside the protoplast – in intercellular spaces (schizogenous ducts) or in dead cells (lysigenous cavities), preventing membrane damage by organic solvents.

Dynamics: formation and utilisation

The synthesis of secondary metabolites is often induced by stress factors: tissue damage, pathogen attack, ultraviolet light, drought (Sunil et al., 2022). For example, when attacked by caterpillars, many plants increase the synthesis of alkaloids and tannins. This is an example of induced defence.

Utilisation of secondary metabolites can occur during cell ageing (anthocyanins are degraded), as well as during the mobilisation of some phenols in lignification processes (cell wall construction). However, most end products (e.g., oxalate crystals or resins) accumulate for life or are removed only with the shedding of organs (leaves, bark).

Agronomic and economic significance

Knowledge of secondary metabolites has enormous applied value:

  1. Medicinal plants: alkaloids, cardiac glycosides (foxglove), flavonoids are the basis of many pharmaceutical preparations.

  2. Spice, aromatic and essential oil crops: mint, coriander, lavender, rose – sources of essential oils for perfumery, the liqueur and spirits industry, and medicine.

  3. Resistance to diseases and pests: breeding for increased content of tannins (resistance to late blight in potato), anthocyanins (resistance to viruses, drought), alkaloids (resistance to aphids). However, excess alkaloids may reduce feed value (e.g., in lupine).

  4. Product quality: anthocyanin content determines the colour of fruits (cherry, grape, currant) and market appeal. In viticulture, the content of phenolic compounds (tannins, anthocyanins) is a key indicator of wine quality.

  5. Allelopathy: the ability of some plants (e.g., walnut, wormwood) to suppress the growth of other crops by releasing phenolic compounds. This is taken into account in crop rotation and choice of neighbours in the garden.

  6. Biotechnology: use of cell cultures to produce valuable secondary metabolites (e.g., taxol from yew, echinacoside from echinacea) without destroying natural populations.

4. Formation, utilisation and intracellular dynamics of cellular inclusions

So far, we have considered cellular inclusions as static structures: starch grains, oil droplets, crystals, vacuoles containing alkaloids. However, in a living cell, everything is in motion. Inclusions are formed, move, change, and, when necessary, are broken down. Understanding this dynamics is critically important for agronomy: it determines how the crop is formed, how seeds accumulate storage substances, how the plant responds to stress, and how product quality changes during storage.

In this section, we will examine the general patterns of biosynthesis, intracellular transport, compartmentation, and mobilisation of ergastic substances, as well as the factors that influence these processes.

4.1. Major pathways of biosynthesis and compartmentation

Cellular inclusions do not form spontaneously in the cytoplasm. Their synthesis is strictly localised in specific organelles, and the finished products either accumulate there or are transported to the site of deposition (usually the vacuole or specialised cavities).

Type of inclusion Site of synthesis Site of accumulation Transport
Starch (assimilation) Chloroplasts Chloroplasts —
Starch (storage) Amyloplasts Amyloplasts —
Storage proteins Rough ER → Golgi apparatus → vesicles Vacuoles (aleurone grains) Vesicular (via Golgi or directly from ER)
Lipids (oils) Smooth ER Oleosomes (in the cytoplasm) Budding from ER
Alkaloids, phenols, glycosides Cytoplasm (enzymes) → transport across tonoplast Vacuoles Tonoplast transporters (ABC, antiporters)
Essential oils, resins Plastids, ER, Golgi apparatus Schizogenous/lysigenous cavities, glandular hairs Vesicular, exocytosis into intercellular space
Calcium oxalate crystals Vacuole (crystal chamber) Vacuole Transport of Ca2+ and oxalic acid across tonoplast

Role of the endomembrane system and vesicular transport

The synthesis of most organic inclusions (proteins, polysaccharides, lipids) is closely linked to the endomembrane system – the endoplasmic reticulum (ER) and Golgi apparatus (Aniento et al., 2022).

  • Proteins: synthesised on the rough ER, then via transition vesicles enter the Golgi apparatus, where they mature (glycosylation, proteolytic removal of signals). From the trans‑Golgi network, vesicles (dense vesicles, clathrin‑coated vesicles) are directed to the vacuole, with which they fuse, releasing their contents (Isayenkov, 2014). In some plants (e.g., wheat, maize), some prolamins can aggregate directly in the ER lumen, forming protein bodies that are then transported to the vacuole bypassing the Golgi (Evert, 2006).

  • Polysaccharides (mucilages, pectins): synthesised in the Golgi apparatus, packaged into vesicles and directed either to the cell wall (exocytosis) or to the vacuole.

  • Lipids: fatty acid synthesis occurs in plastids and the smooth ER. Assembly of triacylglycerols and formation of oleosomes occur on the membranes of the smooth ER. Oleosomes do not have a lipid bilayer membrane but are surrounded by a monolayer of phospholipids and oleosins (Evert, 2006).

Transport across the tonoplast: ABC transporters and antiporters

For water‑soluble secondary metabolites (alkaloids, glycosides, phenols), the final accumulator is the vacuole. Since these compounds are often toxic to the cytoplasm, their transport across the tonoplast is active, energy‑dependent and directed against the concentration gradient.

Two types of transport proteins play a key role (Andreev, 2001; Neuhaus & Trentmann, 2014):

  1. ABC transporters (ATP‑Binding Cassette): use the energy of ATP hydrolysis to directly transfer conjugates (e.g., glutathione conjugates of herbicides, glucosides of flavonoids, chlorophyll catabolites).

  2. Secondary antiporters: use the energy of the proton gradient generated by V‑ATPase and V‑PPase on the tonoplast. These include Na+/H\+-antiporters (for sodium accumulation in halophytes), Ca2+/H\+-antiporters, and alkaloid transporters.

It is important to emphasise that many of these transport systems are induced by stress: treatment with herbicides, pathogen attack, or salinity increases the expression of the corresponding ABC transporters (Andreev, 2001).

4.2. Factors affecting inclusion accumulation

The accumulation of storage and defensive substances is not constant – it varies greatly depending on the developmental stage, nutrient supply, abiotic and biotic stresses.

Genetic and ontogenetic factors

  • Genetic predisposition: this determines whether a crop accumulates starch, oil or protein in its seeds, and in what proportion.

  • Developmental stage: in seeds, active accumulation of storage substances occurs at the end of ontogeny; in leaves, diurnal starch dynamics; in flowers and fruits, accumulation of pigments and sugars as they ripen.

  • Transcriptional regulation: the synthesis of many secondary metabolites is controlled by transcription factors (e.g., MYB factors regulate anthocyanin biosynthesis) (Sunil et al., 2022).

Abiotic factors (light, temperature, water, mineral nutrition)

Light:

  • Required for photosynthesis, and therefore for the formation of primary carbohydrates.

  • UV light and blue light induce the synthesis of anthocyanins (protection against radiation).

  • Light deficiency (shading) reduces starch accumulation in tubers and seeds.

Temperature:

  • The optimal temperature for storage substance synthesis varies among different crops.

  • High temperatures often reduce seed oil content (in sunflower, rapeseed) and accelerate anthocyanin degradation.

  • Low temperatures (autumnal cooling) stimulate anthocyanin accumulation in leaves and fruits (Sunil et al., 2022).

Water regime:

  • Drought increases the synthesis of abscisic acid (ABA), which can accelerate seed ripening and accumulation of storage proteins and oils, but reduces yield.

  • Osmotic stress stimulates the accumulation of compatible osmolytes (proline, sucrose, trehalose) – although these are primary metabolites, they can also accumulate in significant amounts.

Mineral nutrition:

  • Nitrogen: stimulates protein synthesis, but excessive nitrogen may reduce oil content in oilseeds and starch content in potatoes (vegetative growth at the expense of storage).

  • Phosphorus and potassium: necessary for ATP synthesis and sugar transport. Phosphorus is a component of phytin (globoids of aleurone grains). Potassium is a crucial osmotic ion of the vacuole, affecting turgor and sugar transport.

  • Calcium: when in excess, stimulates the formation of calcium oxalate crystals (Khan et al., 2023).

Biotic factors

Attack by herbivores or pathogens:

  • Induces the synthesis of defensive substances: alkaloids, tannins, phytoalexins (phenolic compounds with antimicrobial activity).

  • Enhances the transport of alkaloids into vacuoles (Andreev, 2001).

Symbiosis:

  • In legumes, during nodule formation, nitrogen transport to seeds is enhanced, which may increase storage protein accumulation.

  • Mycorrhizae improve phosphorus nutrition, indirectly affecting ATP synthesis and storage substance accumulation.

4.3. Dynamics: mobilisation and reutilisation of inclusions

Storage substances are not accumulated to remain as dead weight. During seed germination, bud break in spring, or leaf senescence, a phase of active mobilisation sets in.

Hydrolysis of storage substances

  • Starch → amylases, maltase → glucose → export or use in respiration.

  • Storage proteins → proteinases (endopeptidases, aminopeptidases) → peptides and amino acids. In the vacuoles of germinating seeds, a sharp acidification occurs, activating vacuolar proteinases (Isayenkov, 2014).

  • Lipids → lipases → fatty acids → β‑oxidation in glyoxysomes → glyoxylate cycle → carbohydrates (gluconeogenesis) → sucrose (Evert, 2006).

Autophagy and vacuolar degradation

In senescing cells or during starvation, the vacuole can engulf portions of the cytoplasm with organelles – a process of micro‑ and macroautophagy (Andreev, 2001). Vacuolar hydrolases digest these structures, and the monomers are returned to the cytoplasm for reuse.

This mechanism is particularly active:

  • During senile breakdown (autumn leaf senescence).

  • During differentiation of tracheary elements (xylem) – the vacuole destroys the protoplast, leaving only the dead cell wall.

  • During adaptation to starvation (e.g., in cell culture after depletion of sugars in the medium) (Andreev, 2001).

4.4. Interconnections with other cell components

Inclusions are not isolated but interact closely with organelles:

  • Amyloplasts are often surrounded by mitochondria (ready to utilise starch hydrolysis products).

  • Oleosomes during germination adjoin glyoxysomes and mitochondria.

  • Vacuole – the central depot: into it enter ions (via active transporters), organic acids, sugars, pigments. The tonoplast acts as a “second plasmalemma”, regulating exchange between the vacuole and the cytoplasm.

  • Cytoskeleton (microtubules and actin filaments) ensures the movement of vesicles carrying inclusion precursors and maintains the spatial organisation of Golgi stacks, ER and vacuoles (Takatsuka et al., 2023).

Inclusion dynamics are closely linked to the activity of V‑ATPase on the tonoplast: its activity creates a proton motive force for secondary transport of ions, sugars and secondary metabolites (Neuhaus & Trentmann, 2014).

4.5. Agronomic significance of inclusion dynamics

Understanding the processes described above allows the agronomist to:

  1. Manage crop quality through agronomic practices:

    • Application of nitrogen fertilisers during grain filling increases protein content.

    • Limiting nitrogen in late stages of oilseeds increases oil content.

    • Timely irrigation during fruit set and seed filling prevents premature mobilisation of storage substances and yield loss.

  2. Optimise harvest timing: harvesting of potatoes and cereals should be carried out at the stage of maximum starch accumulation, and oilseeds at maximum oil content. Early harvesting leads to a shortfall in dry matter.

  3. Enhance plant resistance: application of micronutrients (silicon, selenium, calcium) can stimulate the accumulation of protective inclusions (silica, calcium oxalate crystals, phenolic compounds), reducing the need for chemical pesticides.

  4. Predict storage life (shelf life): for example, in fruits and tubers, high starch content and low sugar content favour better storage. Knowledge of the dynamics of storage substance conversion helps to develop storage regimes (temperature, humidity).

Thus, cellular inclusions are not a static ballast but a dynamic reserve, governed by a complex regulatory system. An agronomist armed with this knowledge can purposefully influence the formation of a crop with desired properties.

In the next section, we will consider methods for visualisation and identification of cellular inclusions – essential for practical diagnostics in laboratories and field conditions.

5. Methods for visualisation and identification of cellular inclusions

For an agronomist, breeder, or specialist in agricultural product quality, it is not enough to know theoretically which inclusions may be present in a cell. They need to be able to see and identify them – determine the type of starch, detect storage protein, assess oil content, or reveal calcium oxalate crystals. This is necessary for cultivar identification, authentication of raw materials, assessment of maturity and crop quality, as well as for diagnosing metabolic disorders under stress.

In this section, we will consider the main methods used to study cellular inclusions – from classical cytochemical reactions, accessible to any laboratory, to modern microscopic techniques.

5.1. Light microscopy and histochemical reactions

The light microscope is the primary tool of practical plant cytology. To visualise inclusions, fixed and stained material is usually used, and sometimes living preparations. The key principle is selective staining (histochemical reaction): a marker substance (dye) binds to a specific chemical component of the inclusion, making it visible (Evert, 2006; Graham et al., 2014).

Detection of carbohydrate inclusions

Starch:

  • Lugol’s iodine (I2KI): stains starch blue‑violet or black. This is the fastest and most reliable method for identifying starch grains in tissues (tubers, endosperm, cotyledons). Different types of starch (amylose and amylopectin) give shades ranging from blue to red‑violet.

  • Polarisation microscopy (see 5.3): starch grains exhibit birefringence in polarised light and appear as bright objects with a dark “Maltese cross”. This allows even very small grains to be identified without staining.

Inulin:

  • When ethanol (or other alcohols) is added to a section or extract from inulin‑containing tissues, characteristic spherical crystals (spherocrystals) of inulin precipitate. In the living cell, inulin is soluble and invisible; after alcohol fixation, it crystallises.

Detection of lipid inclusions

Fats and oils are hydrophobic and are not stained by water‑soluble dyes. Lipophilic (fat‑soluble) dyes are used to visualise them (Evert, 2006; Mauseth, 2013):

  • Sudan III and Sudan IV: stain lipids orange‑red. The method is simple and reliable. The dye is dissolved in alcohol or propylene glycol, and the section is treated for several minutes.

  • Sudan black B: gives a more intense black‑blue staining, particularly good for revealing small oleosomes.

  • Osmium tetroxide (OsO₄): is reduced by unsaturated fatty acids to a visible black precipitate of osmium dioxide. This is a classic fixative and stain for electron microscopy, but can also be used for light microscopy (Evert, 2006).

Important: as a control, sections can be treated with organic solvents (ether, chloroform, acetone) – lipids are removed and staining disappears.

Detection of storage proteins (aleurone grains)

Proteins can be detected by both general protein reactions and specific ones.

  • Reaction with Sudan III (negative): aleurone grains are not stained by Sudan, distinguishing them from fat droplets.

  • Xylidine blue or bromophenol blue – dyes that specifically bind to proteins (giving blue staining).

  • Millon’s reaction (specific for tyrosine): gives red staining of protein crystalloids. Technically complex, used in research.

  • Biuret reaction (general for peptide bonds): gives violet staining but requires special treatment.

  • Fluorescent methods: staining with aniline blue or fluorescein allows proteins to be visualised under ultraviolet light.

Detection of secondary metabolites

Alkaloids: general alkaloid reagents (Dragendorff’s reagent – potassium iodobismuthate, Mayer’s reagent – mercury iodide solution) give an orange‑red or yellow precipitate with alkaloids in vacuoles. However, these reactions are not sufficiently specific and can give false positives.

Tannins:

  • Iron‑ammonium alum (FeNH4(SO4)2): tannins stain black‑blue (hydrolysable) or black‑green (condensed).

  • Van Iterson’s reagent (vanadate molybdate): stains tannins yellow‑brown.

  • Anthocyanins: water‑soluble pigments, clearly visible in vacuoles of living cells (without staining) depending on pH: in acidic medium – red, neutral – violet, alkaline – blue. To fix the colour, fixation in acidified alcohol is used.

Detection of calcium oxalate crystals and cystoliths

Calcium oxalate crystals are clearly visible under the light microscope without staining, especially in polarised light (they strongly refract light). To clarify the chemical nature:

  • Calcium oxalate is insoluble in acetic acid and dilute hydrochloric acid (unlike calcium carbonate).

  • It dissolves in concentrated sulfuric acid with the formation of needle‑shaped gypsum crystals.

  • Gentle heating of a section in sulfuric acid solution produces gas bubbles (carbon monoxide) only from oxalate.

5.2. Fluorescence microscopy

Many substances possess autofluorescence (intrinsic emission) when irradiated with ultraviolet or blue light:

  • Chlorophyll (not an inclusion, but important) – red fluorescence.

  • Coumarins, phenolic compounds, lignin (in cell walls) – bluish‑white or yellow‑green fluorescence.

  • Alkaloids and some terpenes may also fluoresce, but not always.

Fluorescence microscopy allows rapid localisation of secondary metabolites in tissues without histochemical reactions. Even more informative is confocal laser scanning microscopy (CLSM), which enables three‑dimensional imaging of inclusion distribution.

5.3. Polarisation microscopy

This method is based on the property of some substances (in particular, crystals and ordered polymers) to rotate the plane of polarised light.

Application to inclusions:

  • Starch grains: in polarised light, they appear bright with a characteristic dark cross (Maltese cross). This allows distinction of starch from non‑starch polysaccharides and detection of damaged grains.

  • Calcium oxalate crystals and cystoliths: shine brightly (are anisotropic), clearly visible even against a background of lignified cell walls.

  • Phytoliths (silica): also anisotropic.

The method requires no staining, is rapid, and is indispensable for diagnostics in criminology and seed testing.

5.4. Electron microscopy

To study the ultrastructure of inclusions at the nanoscale, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used (Graham et al., 2014).

TEM allows visualisation of the internal structure of inclusions:

  • Layering of starch grains,

  • Membrane envelope of aleurone grains (tonoplast) and their internal structures (crystalloids, globoids),

  • Oleosomes as electron‑transparent spheres,

  • Crystal lattices of mineral inclusions.

SEM provides a three‑dimensional image of the surface of inclusions after removal of the organic matrix (e.g., calcium oxalate crystals in intercellular spaces, phytoliths).

To determine the elemental composition of crystals, energy‑dispersive X‑ray microanalysis (EDS or EDX) is used, which works in conjunction with SEM. It allows determination of whether a crystal contains calcium, silicon, potassium, or magnesium.

5.5. Practical significance for agronomy

Knowledge of inclusion identification methods is necessary in the following cases:

  1. Cultivar identification and seed testing: the shape of starch grains, type of aleurone grains, and pattern of calcium oxalate crystals are diagnostic features for distinguishing species and even varieties (e.g., in potato, maize, oats).

  2. Assessment of crop quality: starch content (by iodine staining), protein content (by staining), oil content (by Sudan) can be assessed visually or by microphotometry. This is useful for field screening of breeding material.

  3. Authentication of medicinal plant raw materials: the presence of crystals of a specific shape (raphides, druses), crystal type, presence of essential oil cavities and alkaloids are key microscopic features in pharmacopoeias.

  4. Diagnosis of metabolic disorders: for example, accumulation of unusually large calcium oxalate crystals may indicate excess calcium in the soil, while yellowing of vacuoles due to anthocyanins may indicate cold stress or phosphorus deficiency.

  5. Assessment of pasture forages: high phytolith (silica) content in grasses reduces palatability and digestibility, which can be assessed by microscopy of stem and leaf sections.

Thus, microscopic and histochemical methods are not only research tools but also practical means of quality control in crop production, seed production, and the food industry.

In the next section, we will consider the applied significance of cellular inclusions from the perspective of agronomy and biotechnology.

6. Applied significance of cellular inclusions in agronomy

Cellular inclusions are not merely an object of academic interest. Their study underpins many practical aspects of agricultural production: from breeding high‑yielding varieties to developing technologies for crop storage and processing. In this section, we summarise how knowledge of the nature, dynamics and diagnostic methods of ergastic substances helps the agronomist, breeder and technologist solve specific problems.

6.1. Breeding and genetic improvement of crop quality

Traditional breeding and modern biotechnological methods (marker‑assisted selection, genetic engineering) actively use knowledge of the genes that control the synthesis and accumulation of storage substances and secondary metabolites.

Breeding for increased content of target substances

  • Cereal crops: breeding wheat, rice and maize for high protein content (especially gluten) and specific amino acid composition (lysine, methionine) is a priority for the baking and compound feed industries. Markers linked to prolamin and glutelin genes allow elite forms to be selected at early stages.

  • Oilseed crops: increasing oil content and optimising its fatty acid composition (high oleic acid, low erucic and linolenic acids) is the main task in breeding sunflower, rapeseed, soybean and flax.

  • Starch crops: for potato, maize and rice, not only the quantity of starch but also the amylose/amylopectin ratio, size and shape of starch grains (affecting technological properties) are important.

  • Legume crops: increasing the content of storage proteins (globulins) while reducing antinutritional factors (protease inhibitors, oligosaccharides that cause flatulence).

Creating forms with modified secondary metabolite profiles

Reducing undesirable substances:

  • Development of “sweet” (low‑alkaloid) lupin varieties for use of green matter and grain in animal feed.

  • Reducing oxalate content in spinach, rhubarb, beet (for people suffering from urolithiasis).

  • Reducing nitrate and nitrite content (primary metabolites, but hazardous) in vegetables.

Increasing beneficial substances:

  • Breeding for high anthocyanin content (blue, purple varieties of maize, potato, tomato, cabbage) – as antioxidants for functional nutrition (Sunil et al., 2022).

  • Increasing essential oil content in essential oil crops (mint, lavender, coriander).

  • Breeding for high caffeine (tea, coffee), capsaicin (chilli pepper), and other valuable alkaloids.

Genetic engineering of metabolic pathways

Modern methods allow the transfer of genes for the biosynthesis of valuable substances from one organism to another. Examples:

  • Transfer of anthocyanin biosynthesis genes into tomato, rice and maize varieties (producing purple, antioxidant‑rich fruits and grains).

  • Expression of genes for long‑chain polyunsaturated fatty acids (ω‑3) in oilseed crop seeds (e.g., producing “fish oil” in plants).

  • Genetic modification of potato to alter the amylose/amylopectin ratio in favour of amylopectin (waxy starch) or vice versa.

6.2. Plant protection: managing the accumulation of defensive inclusions

Knowledge of the dynamics of defensive substances allows the development of strategies to enhance plant resistance to pests and diseases without chemical treatment.

Induced resistance

Stimulating the synthesis of defensive inclusions by treating plants with elicitors (chitin oligosaccharides, salicylic acid, jasmonic acid) is a promising direction.

  • Treatment of plants (e.g., wheat, tomato, maize) with jasmonate or salicylate enhances the accumulation of alkaloids, tannins, phenolic glycosides and phytoalexins, increasing resistance to aphids, caterpillars, and leaf spot diseases (Andreev, 2001).

  • Application of silicon fertilisers stimulates the deposition of silica (phytoliths) in the cell walls of cereals, creating a mechanical barrier for chewing and sucking insects, and also reduces infection by powdery mildew and rusts (Khan et al., 2023).

Breeding for resistance

  • Varieties with a high capacity to accumulate calcium oxalate crystals (raphides, druses) are often less damaged by slugs, caterpillars and some beetles. Such varieties are promising for organic farming (Khan et al., 2023).

  • Varieties with high anthocyanin content (coloured leaves and stems) often possess increased resistance to viral infections and UV stress.

6.3. Phytoremediation and agroecology

The ability of plants to accumulate and isolate heavy metals in vacuoles and crystals is used to clean contaminated soils.

  • Hyperaccumulators (e.g., some species of mustard, Thlaspi caerulescens) can accumulate in vacuoles up to 3% of dry weight as zinc, cadmium, nickel, binding them with organic acids and phenols (Graham et al., 2014).

  • Growing such plants on contaminated sites (former industrial zones, landfills) and then incinerating them to recover metals is a method of phytoextraction.

  • Some grasses (e.g., miscanthus, reed) accumulate silica and can be used for phytostabilisation (binding) of mobile forms of heavy metals in the soil.

6.4. Biotechnology and industrial use

Cellular inclusions are a source of valuable raw materials.

  • Starch – raw material for bioplastics, adhesives, textile sizes, paper, ethanol.

  • Vegetable oils – not only food products but also raw materials for biodiesel, lubricants, drying oils, soap.

  • Secondary metabolites – the basis of the pharmaceutical industry (alkaloids, cardiac glycosides), food additives (anthocyanins, carotenoids), perfumery (essential oils), biopesticides (pyrethrins, nicotine, rotenone).

Cell cultures allow the production of valuable compounds on an industrial scale without growing whole plants: for example, taxol (anticancer agent) from yew cell cultures, echinacoside from echinacea, anthocyanins and aroma compounds from callus cultures.

6.5. Quality and safety control of products

Microscopic and histochemical methods are actively used to assess the quality and authenticity of agricultural raw materials and food products.

  • Milling industry: the type of starch grains and the structure of the aleurone layer determine the grade of flour (e.g., presence of crushed wheat grains versus rye). Microscopy can reveal admixtures of other cereals.

  • Honey industry: pollen grains (containing specific inclusions) are used to determine the botanical origin of honey.

  • Medicinal plant raw materials: the presence and shape of calcium oxalate crystals, essential oil glands, resin ducts, and type of starch grains are diagnostic features regulated by pharmacopoeias to confirm authenticity.

  • Detection of GMOs: in some cases, modification affects genes controlling starch, oil or anthocyanin synthesis, which can be detected cytologically.

6.6. Predicting storage life (shelf life) and technological properties

  • Potato and fruits: high starch content and low sugar content ensure better storage life. Determining starch content (by iodine staining) is a rapid method when placing produce into storage.

  • Grain: high protein (gluten) and fat content reduces storage life (risk of rancidity, insect infestation). The ratio of storage substances is taken into account when choosing storage and drying regimes.

  • Oilseeds: determination of oil content (by Sudan staining) at early breeding stages allows elimination of low‑value samples.

References

  1. Andreev, I. M., 2001. 'Vacuolar Ion Homeostasis and Detoxification in Higher Plants', Russian Journal of Plant Physiology, 48(5), pp. 673–688.

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