Influence of Environmental Change on Monolignols and the Micromorphology of Leaf Epidermis in Hydrophytes and Terrestrial Plants

Abstract

The review presents data on the role of leaf epidermis, lignin, and its monolignols in the adaptation of higher plants to adverse environmental conditions. It discusses the localization and content of syringyl monolignin and guajacyl monolignin in cell walls and how they affect the elasticity of plant cell walls under different natural conditions. These data are presented using modern methods of laser scanning, confocal microscopy, and scanning electron microscopy. The review also discusses literature data and the results of our own experimental studies on the cellular mechanisms of lignin synthesis and its regulation, as well as the participation of monolignols in plant adaptation to environmental changes. It shows the role of monolignols in regulating water balance and transpiration of plants, as well as in protecting plant cells from abiotic stresses and environmental changes. Recent studies have also shown the influence of lignin on the expression of genes involved in the synthesis of secondary cell walls and metabolites with protective properties. This review indicates the potential for further research into the role of monolignols and confirms that the conservation of species characterized by increased lignin synthesis may be a conceptual basis for the protection and conservation of flora from abiotic stresses.

1. Introduction

Over the past decade, research has shown that the structure and composition of plant epidermis cell walls change in response to various environmental factors. This applies to both terrestrial plants and hydrophytes, which grow in water or on the shore near water. Environmental changes can disrupt cell functions such as growth, water and sun photon absorption, transpiration, strength, and protection. The epidermis of leaves and stems serves as the first barrier between the plant and its environment, protecting it from biotic and abiotic factors such as flooding, extreme temperatures, and pathogen invasion [1, 2, 3]. The leaf epidermis is defined by a combination of structural characteristics and the chemical composition of cell walls, which can change under stress conditions. Trichomes and epidermal wax in the epidermal cells play a role in regulating the absorption of photon energy, decreasing photoinhibition, and reflecting ultraviolet rays. Additionally, the cells of the leaf epidermis play a key role in the development of plants [4, 5, 6]. It is known that one of the main components of the epidermal cell walls, responsible for mechanical strength, leaf elasticity, and water and aqueous solution transport, is lignin and its constituent monolignols. These properties of lignin depend on the content and ratio of its constituent monolignols, which are formed in independent ways [7].

Drought and soil flooding are factors that impact plant growth and species conservation. The study of these environmental factors began with the development of nature ecology and has led to new possibilities for investigating the effects of environmental stress on plant species conservation and vital plant functions. It has been established that the biogenesis and function of leaf epidermis are mediated by the activation of specific enzymes in plants. These studies are described in a series of reviews [7, 8, 9]. Understanding the structural and functional characteristics of leaf epidermis under normal conditions and adapting to stress factors is important. This chapter reviews current knowledge about the structure and function of the epidermis of hydrophytes and terrestrial plants, as well as the participation of lignin and its monolignols under the influence of environmental change.

2. The impact of flooding on leaf epidermis and monolignols

Flooding is a potentially harmful stress for land plants caused by both natural (river flooding, heavy precipitation, and sea tides) and artificial causes (construction of reservoirs and ponds). It can be short-term or long-term, leading to the death of some plant species and crops. Flooding occurs when soils are waterlogged or when irrigated lands are not properly maintained. The combination of changes in salinity and impaired oxygen respiration in the root system inhibits aerobic processes, impairs the absorption of ions from the soil, and affects growth processes. Aerenchyma is formed in the roots, aerobic processes are inhibited, and fermentation begins with the formation of lactic acid, leading to acidification of the environment around the root system [10]. When flooded, plants are affected by reduced lighting, changes in spectrum, a lack of oxygen, and CO₂. Water absorbs light flux, and the attenuation is directly proportional to the water depth. Light is also absorbed by organic and inorganic particles in the water, with the long-wavelength parts of the spectrum being absorbed at greater depths [11].

Underwater plant leaves are impacted by a lack of light and CO₂, which has high solubility but a low diffusion coefficient in fresh water (1.7 × 10⁻⁹ m²/c at 20°C). The pH of fresh water also affects gas solubility and the ratio of CO₂ to O₂ in water [12]. The air contains 0.03% carbon dioxide, 78.09% nitrogen, and 20.95% oxygen. In water, the amount of dissolved oxygen depends on atmospheric pressure, with higher pressure leading to greater solubility of oxygen. At 1 atm and 25°C, 0.023 g of oxygen is dissolved in 1 kg of water. The solubility of carbon dioxide in water is 200 times higher than that of oxygen [11]. The CO₂ content in fresh water is 350 mM m⁻³, and the diffusion of gases in water is also lower. Underwater plants require 30 times more free CO₂ to saturate their leaves [12] and therefore use CO₂ ions as a carbon source [13]. Growth conditions in aquatic environments can cause hydrophytes to die when light is limited, and oxygen levels in the water and in plants decrease, leading to the inhibition of carbohydrate metabolism [14].

These adaptations allow hydrophytes to efficiently carry out photosynthesis and obtain the nutrients they need despite the challenging conditions. Additionally, some hydrophytes have specialized air channels and aerenchyma tissue that facilitate gas exchange and help them survive in waterlogged environments. These adaptations are essential for the survival of hydrophytes and marsh plants in their unique habitats [2, 15].

The features aid in acclimatization and support plant resistance to underwater existence, allowing for CO₂ to enter photosynthetic cells through diffusion in the outer epidermal walls. The structure of cell walls in underwater leaves and stems differs from that of surface hydrophytes and mesophyte leaves, highlighting the importance of comparative structural and functional studies in understanding plant adaptation to aquatic environments.

2.1 The submerged leaf еpidermis structure

The outer cell walls of the epidermis and leaf stomata act as the first barrier and transport route for CO₂ and water, as well as the point of contact with the environment for plant organs. The cuticle of mesophyte leaves’ epidermal walls contains pores that facilitate cuticular transpiration and water absorption. The functionality of cuticular pores in aerial leaves is influenced by the temperature and humidity of the surrounding air [16, 17]. In flooded plants, the cuticle is also synthesized in the epidermis and deposited in the periclinal cuticle sheaths. In certain species of higher aquatic plants (Menta aquatica, Oenanthe aquatica, and Rumex palustris) that are tolerant to complete flooding, old aerial leaves often die off underwater, giving way to new dissected leaves with a different cell structure [2, 18, 19] and altered cuticle structure [20, 21], up to and including its hydrolysis [22].

Aquatic plants, also known as hydrophytes, have a super hydrophobic cuticle on the epidermis of their leaves and stems. This prevents the formation of a water film on the surface of submerged organs, which leads to normal gas exchange in the leaves and to photosynthesis [23, 24]. Recent studies have focused on the structure of the cuticle of epidermal cells in underwater plants, particularly. The presence of cuticular pores in the cell walls of the epidermis of underwater leaves and the functioning of the epidermal cell wall were studied [15, 25, 26].

The study of the ultrastructural peculiarities of wall epidermis in submerged leaves of Myriophyllum spicatum, Potamogeton perfoliatus, and Potamogeton pectinatus water plants showed the presence of a very thin cuticle layer with pores of various sizes and densities (Figure 1) [25, 27].

Figure 1.

Scanning electron microscopy of the epidermal cells of the submersed leaves of Myriophyllum spicatum (a, b, c, d), Potamogeton pectinatus (e, f, g, g′), and P. perfoliatus (h, I, j). Cuticular pores are indicated by arrows in figures a, d, g, g′, and j. Abbreviations: C, cuticle; Cw, cell wall; Ch, chloroplast. Scale bars = 10 μm (a, e, h); = 5 μm; = 1 μm (b, c, f, i); = 100 nm (d, g, g′, j), [25].

The surface of the leaf epidermis of M. spicatum was covered with a continuous, uneven layer of cuticle, and individual epidermal cells were not distinguishable; chloroplasts were in epidermis cells (Figure 1b). In the epidermis of M. spicatum, cuticular pores were in a wavy, round, or straight form (Figure 1c and d). Their length was up to 130 nm, the diameter of the pore was 20 ± 4 nm, and the density of pores was 12–15 per 1 mm of cuticle extension. The epidermis of P. pectinatus leaves covered with almost parallel cuticle ridges (Figure 1e–g′), the height of which varied from 1.5 to 5 μm and the length from 12 to 45 μm. Due to the continuous cuticle layer, the boundaries of individual cells were not distinguishable. In contrast, the epidermal cells of P. perfoliatus leaves (Figure 1b) had clear walls. The walls of the epidermis in P. perfoliatus were two times higher in the wall and five to seven times the thickness of the periplasmic layer, and the thickness of the cuticle decreased. The cuticle pores were faintly marked; the average size of the pores was about 4–5 nm. In contrast, the outer wall of the epidermis of P. pectinatus was very wide (up to 5 μm). Cuticle pores were located in the outer layer of the wall (Figure 1f and j); the average diameter of the pore was 17 ± 3 nm, and the length was 80–100 nm. The density of pores was 20 per 1 μm of cuticle extension. The authors suggest that the presence of cuticle pores in the walls of the epidermis of M. spicatum, P. perfoliatus, and P. pectinatus is a morphological sign of the adaptation of these species to submerged conditions. It is known that a very thick cuticle is characteristic only of mesophytes and xerophytes, and the cuticle pores of walls are very narrow [17]. The functioning of cuticle pores depends on endogenous and exogenous factors, including leaf age, air moisture, and air temperature [28]. The cuticle of the outer cell wall of the submerged leaf epidermis is involved in the transport of CO₂ and HCO₃ from water into the apoplast, then to the protoplast for photosynthesis [2].

2.2 Monolignols in submergenced leaves of hydrophytes

This section will discuss the presence of monolignols in the leaves of hydrophytes that are subjected to flooding. Lignin is essential for plant adaptation to stress, especially in response to flooding and siltation. It is a polymer of aromatic alcohols synthesized in cell walls that have completed stretching growth. Lignin helps make cell walls impermeable to water and aqueous solutions, forming a barrier to pathogens in the epidermis. It is composed of monolignins formed from p-hydroxyphenyl, guajacyl, and syringyl components, which differ in the degree of methoxylation [29]. Flooding and siltation impact the lignification of cell walls, with flooding being the main effect and mechanism of adaptation of the rice root system due to a lack of oxygen. The study investigated the impact of 50 cm deep flooding on the lignification of rice stems by comparing three rice varieties: flood-resistant (FR13A), flood-sensitive (IR42), and flood-adapted (Sabita). The flood-resistant mutant, FR13A, carries the genes SUB1A and SUB1B on the ninth chromosome, which encode transcription factors responsible for ethylene synthesis [30]. When subjected to flooding, the flood-sensitive variety IR42 showed a significant increase in stem growth rate and leaf tensile strength compared to the flood-resistant variety. A negative correlation was found between stem elongation, lignin content, and the activities of coniferol alcohol dehydrogenase, and phenylalanine ammonium lyase. Additionally, a negative relationship between plant height and the content of structural polysaccharides (cellulose and hemicelluloses) was also observed. This suggests that flood-sensitive rice plants may have a different mechanism for coping with flooding stress compared to flood-resistant varieties.

According to researchers [31, 32], underwater (submerged) plant organs experience stress from the weight of the water column and the mechanical action of waves, leading to tension in flooded organs. The effect of this stress on the structure and functioning of cell walls in flooded plants are still not fully understood. While there is data on the impact of stress on the cell walls of land plants, the specific effects on flooded plants are not well documented. For example, in naked-seeded plants, the wood stress response occurs at the level of several vessel elements that have a small diameter, thick cell walls, high cellulose content, and low lignin [33]. These walls are characterized by a small amount of guajacol, a high content of syringin, and a three-layer structure [34].

A study examined the presence of lignin and monolignols in the leaves of the hydrophytes Myriophyllum spicatum, Potamogeton pectinatus, and P. perfoliatus (Figure 2) [35, 36]. Cytochemical analysis of monolignols in different tissues of submerged leaves of these three species revealed a higher content of syringyl and guajacyl in the epidermis cell walls compared to the walls of the photosynthesizing parenchyma. The study showed that the cell walls of underwater leaves of M. spicatum contained syringyl and guajacyl components, which emitted blue and green fluorescence, respectively, when combined with diphenyl boric acid-2-aminoethyl ester (DPBA) (Figure 2b–d). High levels of both guajacyl and syringyl were found in the cell walls of vessels and the anticlinal walls of the epidermis, while in the cell walls of the parenchyma, these levels were almost half as low. It is worth noting that the fluorescence of monolignins was significantly higher in the corners of the parenchymal cells compared to along the walls. Additionally, the ratio of syringyl to guajacyl content in the walls of photosynthetic parenchyma was quite high, especially in the first layer of photosynthetic parenchyma. In terms of the S/G ratio in leaf M. spicatum, cells are arranged in the following order: epidermis > vessels > parenchyma. Whereas in other species—P. pectinatus, this S/G ratio was in different order: parenchyma > epidermis > vessels > walls in the corners of parenchyma [36]. The high levels of monolignins in the corners of epidermal cells suggest an increase in the strength of these cells, making it difficult for pathogens to invade [37]. This epidermis also act as a barrier and protection of the leaf from the action of ultraviolet, which provokes the intensified lignin synthesis [38].

Figure 2.

The general view of underwater leaves of Myriophyllum spicatum and Potamogeton perfoliatus and the cytochemical fluorescence of monolignols bound to specific indicators diphenyl boric acid-2-aminoethyl ester (DPBA) in the leaves of M. spicatum leaves and P. perfoliatus was visualized with confocal laser scanning microscopy. Syringyl has blue fluorescence; guajacyl has green fluorescence; chlorophyll has red fluorescence. The histogram in figure e shows the fluorescence intensity of guajacyl, syringyl, and chlorophyll autofluorescence. The abscissa represents the distance (μm) scanned in figure d, which is shown by the white line. Scale bars: 100 μm [35].

The presence and distribution of lignin in the tissues of underwater and above-water leaves of the hydrophyte Sagittaria sagittifolia, as well as the increased lignin content in submerged leaves, are similar to the data on the increase in lignin in flooded stems of Ludviga repens [39]. Little demonstrated that flooding L. repens stems increases the lignin content by 1.6 times compared to stems that are not flooded [39]. And this is what helps the plant to keep the flooded stem upright. Submerged leaves lack trichomes, stomata, and thick cuticles [15], making the surface of plant organs submerged in water more sensitive to pathogen invasion. Leaves and stems synthesize lignin in response to pathogen attack [37]. Exogenous and endogenous factors cause increased lignin synthesis in the underwater leaves of the studied species of water nut and arrowroot. Submerged plant organs must withstand water pressure and wave action, which affect the growth and structural and functional parameters of tissues, cells, and their walls [32, 40]. Lignification in the cell walls of parenchymal cells and vessels in mesophytes begins in the corners of cells and in the middle plate, which is filled with pectin substances. These zones become enriched with hydroxyphenyl and guajacol units [32, 41], as well as phenols such as coumaric and ferulic acids, which act as a foundation for lignin synthesis [42, 43].

2.3 Floating and above-water leaves

The epidermal structure of floating leaves is adapted to their aquatic environment. These leaves have a thick waxy cuticle and a reduced number of stomata, which helps to minimize water loss. Additionally, the epidermal cells may contain air spaces to provide buoyancy.

The micromorphology and ultrastructure of the epidermis of floating leaves of Hydrocharis morsus-ranae showed some differences from those of submerged leaves. The floating leaves of H. morsus-ranae are hypostomal, with differences in the ultrastructure and size of the main cells of the upper and lower epidermis. The upper epidermis cells are polygonal, oval, occasionally rounded, and tightly adjacent to each other (Figure 3a).

Figure 3.

The scanning electron microscopy images display the adaxial (a) and abaxial (b) surfaces of Hydrocharis morsus-ranae leaves. The fluorescence of syringyl and guajacyl is visualized with laser confocal microscopy on the adaxial (c and e) and abaxial (d, g) surfaces of the leaves and it is also shown on histograms (f and h). Abbreviations: An—anticlinal wall, pe—periclinal wall, and St—stomata. Scale bars: A, and b = 10 μm, c = 50 μm, and d = 100 μm.

The anticlinal walls are elevated above the periclinal walls, forming a cup-shaped depression in each cell. Stomata are located on the lower epidermis in no particular order (Figure 3b), with an average density of 33.1 ± 1.7 per 1 μm² of area. They are of the parasitic type, with accompanying cells located along the guard cells. The usual epidermal cells of the lower epidermis, similarly to those of the upper epidermis, form cushion-like depressions. The epidermal cells vary in shape, with round cells having a diameter of about 10 ± 1.2 μm and oval cells having a long axis ranging from 10 ± 0.7 to 37 ± 2.3 μm and a short axis from 7 ± 0.3 to 22 ± 1.9 μm, respectively. The anticlinal walls are wavy, reaching a height of 7 ± 0.4 μm. Periclinal walls are concave within the leaf blade and are located in depressions. Using the Pascal program (LSM), it has been calculated that there is air under the lower surface of the leaf, in the recesses of the main epidermal cells, with a content of 119,700 ± 2300 μm³ on an area of 1 cm². Aerenchyma was also revealed between cells of the mesophyll and epidermis. It was established that the leaves of H. morsus-ranae have slightly deeper stomata in the epidermis, which is situated on the lower leaf surface. The presence of stomata on the lower epidermis of H. morsus-ranae was first described by Ancibor [44]. This investigator noted stomata deepened placement in the epidermis to increase the buoyancy of the leaves and not only for gas exchange. The author writes only words about the complete absence of lignification of the leaves of the toadstool or its very low content (although this author’s figures are not shown), which ensures the flexibility of organs in water.

Our investigation found that the air content under the lower surface of the flooding leaf of H. morsus-ranae was 119,700 ± 2300 μm³ per 1 cm². We also observed differences in the height of the anticlinal walls on the upper and lower surfaces of the leaves. The height of the anticlinal wall in the upper epidermis was one and a half times larger than that of the cells of the lower epidermis, and its height was 7 ± 0.4 μm. Similar protrusions of anticlinal walls were found in the surface and submerged leaves of S. sagittifolia [45]. Leaves of free-floating hydrophyte species, such as Anubias barteri, Hydrophila odora, Bacopa caroliniana, and Hydrocharis sp., characterized by presence of aerenchyma in leaf petioles and pedicels [46], and also by poorly developed mechanical and conductive tissue in leaf [47]. Aerenchyma was also detected in the stems of species collected in South Korea [48] and in the Nelumbo nucifera peltate leaf [49].

We demonstrated that syringyl and guajacyl monolignols, when combined with the specific fluorescent indicator diphenyl boric acid-2-aminoethyl ester, exhibited bright blue and green fluorescence in the periclinal and anticlinal walls of adaxial and abaxial epidermal cells of Hydrocharis morsus-ranae leaves (Figure 3c–h). It was shown that the intensity profile of DPBA-syringyl and DPBA-guajacyl complexes varied depending on the surface of the epidermis, the cell type. The anticlinal walls of usual cells in both epidermis, and the walls of stomata are characterized by highest fluorescence intensity. The intensity profile of syringyl in the periclinal and anticlinal walls of usual cells of adaxial surface was 27 ± 1.1 and 39 ± 1.3 relative units, respectively. On the abaxial surface, it was 91 ± 7.2 and 190 ± 11 relative units, respectively. The intensity profile of guajacyl was higher than that of syringyl in walls, measuring 53 ± 4.1 in the periclinal wall and 118 ± 13 relative units in the anticlinal wall of usual cells in the adaxial epidermis. The intensity profile of guaiacyl in such walls of the abaxial epidermis was significant in stomata, periclinal, and anticlinal walls, measuring 100 ± 10 and 120 ± 11 relative units, respectively, and also in the anticlinal wall of typical cells, measuring 91 ± 7.1 relative units. The intensity of guaiacyl in the periclinal wall of typical epidermal cells did not differ from that on the adaxial surface, measuring 115 ± 10.1 units.

Previous research has shown that an increase in the quantity of syringyl/guajacyl (S/G) acts as a chemical barrier, enhancing cell defense against water penetration and pathogens [50] while also increasing the mechanical durability of cells [51]. Based on our findings, we hypothesize that the elevated content of syringyl in the abaxial epidermis of the studied species can enhance the mechanical properties of the cell walls. Interestingly, the content of guajacyl was found to be similar in the upper and lower epidermal walls. Given the known function of guajacyl in wall softening, it is plausible that its high content in the cell walls of floating leaves of H. morsus-ranae promotes optimal movement of the leaves and the plant itself under the influence of water current waves.

S. sagittifolia and Trapa natans are convenient for studying the structure of above-water leaves of hydrophytes [26, 36, 45, 52, 53]. The ultrastructure of epidermal and mesophyll cells of the leaves of S. sagittifolia shows differences in cell wall structure between above-water and submerged leaves (see Figure 4a–d). The outer walls of the adaxial epidermis cells of above-water leaves of S. sagittifolia were about 1.5 μm wide and up to 2.5 μm in the area of the anticlinal walls, characterized by layering. The mesophyll cell walls are thin and have a typical pecto-cellulose structure. A similar thinning of the epidermal cell walls was found in the free-floating leaves of T. natans [53].

Figure 4.

Fragments of epidermal cells of surface above-water (a, c, d) and submerged (b) leaves of Sagittaria sagittifolia (a and b) and Trapa natans (c and d). In submerged leaves, the epidermal cell walls are thinner and loosened. Figures e, f, g, h, and i show the cytochemical fluorescence of lignin in cells of the above-water S. sagittifolia. Lignin has a yellow fluorescence: e, f, adaxial epidermis; g, h, i, abaxial epidermis. In figure i, the histogram of intensity lignin yellow is presented. Figure e shows 3D structure. Scale bars: а, b, c, d = 1 μm; e, f, g = 50 μm [26].

The mechanism of cell wall thinning and loosening, particularly in hydrophytes, has not been fully understood. There are several proposed models for cell wall loosening, including associations with decreased cell turgor, reduced activity of polysaccharide synthetases [54], apoplast acidification [55], and activation of phytohormones [56, 57]. It is revealed that phytohormones induce stretching and increase wall plasticity, depending on the apoplast pH [54, 56]. Additionally, phytohormones act as effectors of wall loosening. It has been established that one mechanism of cell wall loosening in higher plant stems is the participation of brassinosteroids, which regulate the expression of genes for loosening proteins, namely xyloglucan endotransglucosylases, and act at the transcriptional level [57].

Studies of lignin in the surface leaves of S. sagittifolia and Trapa natans showed that cells of the upper and lower epidermis contain lignin, with different relative content in the periclinal and anticlinal epidermal walls [53, 58]. Laser confocal microscopy revealed light yellow fluorescence of lignin in the epidermal and mesophyll cell walls, clearly visible in the leaf cells of the arrowhead leaves of S. sagittifolia (Figure 4c–f). The analysis showed that the relative lignin content in the walls of the upper epidermis was higher than in the walls of the lower epidermis. Additionally, the relative lignin content in the mesophyll cell walls was two to four times lower than in the walls of the upper epidermis. Biochemical analysis showed that the total lignin content in above-water leaves of S. sagittifolia was 26.5 ± 3.1 mg/g dry weight, whereas in the underwater leaves, its content was more than twice as high at 58.6 ± 4.9 mg/g dry weight.

The presence and distribution of lignin in the tissues of underwater and above-water leaves of the hydrophyte S. sagittifolia, as well as the increased lignin content in underwater leaves, are similar to the findings on the increase in lignin in flooded stems of Ludviga repens [39]. This phenomenon is attributed to the complex cycle of lignin synthesis, which is influenced by various endogenous and exogenous factors such as phytohormones, light, temperature, ambient gas concentration, and biotic stresses [37, 59]. Lignin provides mechanical support to plant tissues, enabling them to remain upright and withstand water pressure and wave action [31, 32]. Submerged plant organs must adapt to these environmental challenges, which affect their growth and structural parameters. Additionally, submerged leaves lack trichomes, stomata, and thick cuticles, making them more susceptible to pathogen invasion [25, 52]. In response to pathogen attacks, leaves and stems synthesize lignin as a defense mechanism [37]. Therefore, both exogenous and endogenous factors contribute to the increased lignin synthesis in the underwater leaves of the studied species of water nut and arrowroot.

A cytochemical analysis of S. sagittifolia revealed the presence of syringol and guajacyl in the cell walls of the upper and lower epidermis, palisade, and spongy parenchyma, as well as in the walls of the vessels of the conducting bundles (Figure 5) [45, 58].

Figure 5.

The general view of the above-water (a) and submerged (g) leaves of Sagittaria sagittifolia. The cytochemical fluorescence of lignin components in the leaves is as follows: DPBA + syringyl complex fluoresces blue (b, d, h); DPBA + guajacyl complex fluoresces green (b, c, e, i); chlorophyll autofluorescence is red: b, h—syringyl + guajacyl; c, e, i—guajacyl; d—syringyl. Figure e, f shows the histogram of guajacyl fluorescence intensity (green line) and chlorophyll autofluorescence (red line). The fluorescence intensity is in relative units. The abscissa represents the distance (μm) scanned in figure e, which is shown by the white line. Scale bars: 100 μm (c) and 50 μm (b, d, h, i) [58].

The relative content of syringyl and guajacyl varied in the walls of the studied tissues. The highest values of guajacyl are found in the epidermis, palisade parenchyma, and vessels. The ratio of syringyl to guajacyl content (S/G) was highest in the abaxial epidermis, followed by the adaxial epidermis, spongy parenchyma, palisade parenchyma, and vessels. Notably, the periclinal walls had higher S/G values compared to the anticlinal walls, regardless of the type of epidermis. An increase in the S/G ratio strengthens the chemical barrier to protect the cell from water penetration and pathogen invasion [50]. Additionally, the S/G ratio indicates an increase in the mechanical strength of cell walls [51]. Leaves immersed in water are constantly in contact with surrounding aquatic microflora and numerous algae, and they can withstand constant water pressure and wave action [15]. Both the periclinal and anticlinal epidermal walls of underwater leaves protect the leaf cell surface from the exogenous effects of the aquatic environment by regulating the synthesis of lignin components.

3. Effect of drought on epidermis structure of plant leaves and monolignols content

Environmental stressors such as drought, salinity, salt fog, solar radiation, high light intensity, low nutrient availability, and high soil temperature affect the structure of a plant’s leaves [60, 61]. These changes can impact the morphology of the leaves, which is directly regulated by primary stress signals that trigger mechanisms for adaptation, crucial for plant survival in stressful habitats [62]. Drought, for example, can lead to a loss of leaf turgor, which is the plant’s ability to maintain cell turgor pressure during dehydration and is a strong predictor of the plant’s response to drought [63]. Various factors, including structural and functional changes in both the roots and leaves, help preserve leaf turgor, particularly in the leaf structure. Drought stress can reduce water potential and turgor, leading to inhibited photosynthesis, metabolism, and potential cell death [64]. Lignin plays a role in maintaining tissue water potential by regulating cuticular and transcriptional transpiration, thus reducing water consumption [65]. Vegetation in coastal sand dunes faces environmental stress such as drought, salinization, solar radiation, high light intensity, low nutrient availability, high temperatures, sand instability, and sea or river breezes [60, 61, 66]. These stressors affect the structure of plant organs [67].

Drought causes changes in leaf structure and plays a significant role in leaf adaptation. Specific patterns in leaf morphology are regulated by primary stress signals, triggering mechanisms of structural and functional adaptation crucial for plant survival in stressful habitats [62]. Structural adaptations to drought conditions include water-storing parenchyma leaf blades, curling, deepening of stomata, and leaf pubescence. These adaptations help psammophytes maintain optimal water balance in plants. A study of 54 taxa in the Boraginaceae family found mineralized structures at the base of leaf trichomes in psammophytic species, as well as the presence of calcium and potassium ions [68].

For example, a study of species specific to sand dunes (Marina di Vecchiano coast, Italy), namely Calystegia soldanella, Euphorbia paralias, and Otanthus maritimus, showed that the leaves of C. soldanella and E. paralias had a flat shape, while those of O. maritimus were rolled up. The surface of the studied leaves of the three species was covered with a dense layer of glandular multicellular trichomes, which were located both on the entire surface and on the edges of the leaves [66, 69, 70]. Phragmites australis (Cav.) Trin. ex Steud is a plant species that can thrive in both water and drought conditions. It has a wide geographical distribution and is used in agriculture, construction, and energy technologies. The structure of the leaf epidermis of P. australis is a typical example of a plant that can grow in various environments. For terrestrial plants of this species, the leaf epidermis has two distinct zones: the stomata zone and the convex vault zone without stomata. The adaxial surface of the convex vault and stomata zone is covered with wax (Figure 6) [71]. The width of the vault zone ranges from 44.2 ± 3.1 to 65.0 ± 5.9 μm, while the width of the stomata zone ranges from 67 ± 4.1 to 100 ± 9.3 μm. Trichomes are present in the stomata zone, with a density of 376 ± 15 per mm2 area. The stomata are elongated-oval, with a density of 450 ± 39 on adaxial surfaces and 699 ± 53 per mm² area on abaxial surface, accordingly. The periclinal walls of guard cells are covered with wax granules. The abaxial epidermis also has a vault and a stomata zone. The widths of the stomata zone range from 50 ± 3.7 to 120 ± 10.1 μm, and the width of the convex vault zone is 53 ± 4.7 μm. Nipple-like short cells are present in the vault zone, covered with a thick layer of cuticle and wax structures. Guard cells and surrounding cells of stomata are encrusted with wax depositions. This investigation highlights the differences in leaf micromorphology and in the epidermis ultrastructure in the leaves of P. australis grown in different environments (in the water along the shore and on land, far from the water’s edge) [71]. The author revealed that the density of stomata and trichomes in the leaf epidermis of terrestrial plants comparably was significantly lower than that in the air-water ecotype of this species. Similar changes in stomata density have also been found in rice leaves [72].

Figure 6.

The general view of Phragmites australis plants growing in terrestrial soil (a); the ultrastructure of the leaf adaxial (b) and abaxial surfaces (c). Micrographs of fluorescence of monolignols in leaf epidermis of P. australis are also presented, with syringyl exhibiting blue fluorescence and guajacyl exhibiting green fluorescence, while chlorophyll exhibits red autofluorescence. Monolignols are shown in figures d, e, f, and g, with syringyl and guajacyl in walls. Figures f and f’ present a histogram of the fluorescence intensity of syringyl (blue line), guajacyl (green line), and chlorophyll autofluorescence intensity (red line), while figures g and g’ present a histogram of the fluorescence intensity of guajacyl and chlorophyll. The ordinate represents fluorescence intensity in relative units, while the abscissa represents the distance (μm) scanned on figures f and g. This distance is shown as a white line on figures f and g. Figures d and f represent the adaxial epidermis, while figures g and g′ represent the abaxial epidermis. Scale bars: 100 μm (figures b and c) and 200 μm (figures d and e) [71].

The study of both surfaces of the leaves of Corynephorus canescens grown in drought conditions showed the presence of non-glandular trichomes and wax. These trichomes protect the aboveground organs of plants from attack by sucking insects and herbivores, as well as from mechanical damage [73]. Meanwhile, the epicuticular wax, which is found on the cuticle surface, plays a crucial role in structuring the surface at a sub-cellular scale. It self-assembles into crystals that act as the main barrier to water and small molecule loss from the cell, as well as reducing the uptake of liquids and molecules from the outside [74, 75]. The presence of wax and trichomes in the epidermal cells is an adaptive feature of heliophytes and psammophytes that grow in soil under drought conditions. Psammophytes, which grow on sand dunes, have developed mechanisms to resist and adapt to environmental variations [76, 77, 78, 79]. Structural features of the epidermis of psammophytes include twisted leaf blades, changes in stomatal density, increased indumentum, thickened cuticles, and the synthesis of calcium inclusions in cells and intercellular spaces, which help maintain the optimal water balance of such plants during of droughts [80, 81, 82].

Trichome density also increases during drought [83]. A dense coating of trichomes changes the optical properties of the leaf surface and can reflect or absorb a certain wavelength of light. Trichomes, which capture a layer of air on the leaf surface, can also preserve heat and moisture [73]. In the cells of the epidermis and hypodermis of Corynephorus canescens leaves, we found inclusions of various shapes and sizes containing calcium and potassium. These inclusions can also be of different nature, as reported in the literature [69, 84].

The study of the structural and functional characteristics of Phragmites australis grown under drought conditions showed that such stress causes increased lignin synthesis [71].

A cytochemical and laser confocal microscopic study of monolignols in the leaf epidermis of the air-water and terrestrial ecotypes of Ph. australis revealed that a decrease in soil moisture during moderate drought led to an increase in monolignols content and an increase in the S/G ratio. We found that trichomes and the cells of the vault zone in the leaf epidermis are the main accumulators of monolignols. We hypothesize that the increase in monolignol content in the epidermal cells of the adaxial and abaxial leaves of terrestrial plants leads to a reduction in cuticular and stomatal transpiration, optimizing the water balance of plants grown in moderate drought (Figure 6d–g). It was established that the intensity of syringyl fluorescence in terrestrial plants was 1.22 times higher in the upper epidermis and 2.4 times higher in the lower epidermis compared to air-water P. australis plants. Similarly, the distribution intensity of fluorescence guajacyl was significantly higher in terrestrial plants, being nine times higher on the adaxial surface and 8.3 times higher on the lower epidermis compared to air-water plants. This phenomenon could be explained by the different environmental conditions experienced by terrestrial and air-water plants, which may lead to variations in the composition and distribution of lignin components.

The differences in the relative content of monolignols in different epidermal cells of Phragmites australis leaves can be explained by the involvement of peroxidase and laccase enzymes in lignin synthesis, which are key enzymes in the polymerization of lignin monomers [85]. Plant protection during drought occurs at the cell and molecular levels, including the increase of enzyme activity of CoA reductase and cinnamyl alcohol dehydrogenase, as well as the expression of relevant genes [86]. Based on these findings and our results, we can infer that the enhanced synthesis of monolignols (S and G) in the leaves of terrestrial P. australis plants is due to the activation of these enzymes.

It is known that the increase in lignin causes osmotic stress, leading to water loss or cell death and inhibiting plant growth and development [64]. Lignin can decrease water flow by cells, supporting osmotic balance and cell integrity [87, 88, 89]. Considering the literature and our results on the increased S/G ratio in the cells of conductive vessels and cells of the upper epidermis in leaves of the terrestrial ecotype of P. australis, we assume that the main factor influencing this indicator is relative drought and soil moisture. Previous experiments have shown that an increase in S/G quantity acts as a chemical barrier, intensifying cell defense from water penetration and pathogens [50] and increasing the mechanical durability of cells [51]. It is likely that the increased S/G value in the stomata and trichome of the stomata zone in aerial-aquatic and terrestrial reed plants increased the mechanical properties of the leaf epidermis of the studied species.

According to the literature, drought, similar to soil salinity, has various negative effects on plant growth, such as osmotic stress and inhibition of organ growth [64, 87, 88, 89]. However, plants can adapt to unfavorable conditions by exhibiting phenotypic plasticity, which includes an increase in lignin synthesis and changes in leaf blade shape (twisting into tubes or folding) [90, 91, 92]. This helps the plant maintain optimal water balance [38, 89] and protects it from UV radiation. Lignin acts as an “aggressive” wall component that can displace other polysaccharides [93]. Researchers have proposed a biomechanical model demonstrating that crystalline cellulose is replaced by lignin in the stomatal end walls, serving to strengthen the wall.

Besides, lignin and its monomers are a chemical barrier that increases the protecting of cells from penetration of water and invasion by pathogens [50], and the sign (S/G) testifies to the increase of mechanical durability of cells [51]. The presence of lignin and crystalline cellulose in guard cells implies different biomechanical functions. Our experimental data suggests that in terrestrial plants, leaves of reeds receive a higher dose of ultraviolet radiation than leaves of reeds in plants growing in water. The high content of syringyl monolignol in terrestrial plants can serve as a marker for making strong bricks, while the increased content of other guajacyl monolignol in air-water plants can be a marker for getting soft paper from the reeds. The presence and ratio of monolignols in the cell walls of the epidermis and vessels correlate with early-received data for upland plants [94]. It is possible that the leaf cell walls of the plants studied also contain p-hydroxyphenyl, which plays a substantial role in lignin structure and can be determined using UV spectroscopy [95].

Thus, it was established that the changes at tissue and cellular levels are accompanied by an increase in lignin and cellulose in plants grown under drought stress. However, changes in lignin content in plants under the influence of the environment vary, with some experiments showing a decrease and others showing an increase. This may depend on the plant species and growth phase used for the study. Plant protection during drought occurs at both the cellular and molecular levels. Researchers have noted increased enzyme activity of CoA reductase and the expression of relevant genes in response to moderate water deficiency in the soil. This led to an increased synthesis of monolignols in the cell walls of P. australis leaf. The increase in syringyl and guajacyl content in the cell walls of epidermis cells of terrestrial plants may reduce cuticular and stomata transpiration, optimizing the water balance of plants grown in moderate drought and increasing plant resistance to drought.

4. The influence of cold on leaf lignin

The main causes of plant death from cold are direct or prolonged exposure to low temperatures and the impact on crop growth and development. Researchers categorize plants into three types based on their response to cold: sensitive to cooling, resistant to cold, and adapted to low temperatures [96]. Plants that overwinter in temperate climates can tolerate the freezing of a significant amount of water on the cell surface. Ice crystal formation begins when exposed to temperatures ranging from −1 to −3°C for one hour. The receptors for cold perception in a plant cell have not yet been identified, but it is assumed that the cold signal is perceived by converting it into a calcium signal, which triggers further processes [97].

A study of the structural and functional features of plants growing in the Arctic may provide an answer to the natural mechanisms of adaptation of the plant world to the effects of low temperatures. Polar plants are characterized by the inhibition of respiration in the light, with ATP production occurring during the day through photorespiration and dark respiration observed only at night. This has been shown in polar species such as Betula nana L. (dwarf birch) and Salix pulchra (tea vine) [98].

Cold shock proteins and genes, including desaturases, and dehydrins are hydrophilic and are secreted into the cell walls of epidermal tissues. They are then placed on the surface of ice crystals, inhibiting their further growth [99, 100]. Freezing water from the outside of the cell prevents the formation of ice crystals in the protoplast but leads to dehydration of the cell’s macromolecular structures. This occurs due to increased outflow of water from the cell through the plasmalemma, resulting in changes to the composition of phospholipids. Water then goes into the intercellular spaces, causing severe dehydration of the protoplast [101, 102]. These events lead to a decrease in cell volume, deformation, and changes in the mechanical properties of cell walls [103, 104, 105].

Cold acclimatization leads to changes in the structure of the cell wall and the composition of polysaccharides, including thickening of the cell wall of the epidermis and alterations in the synthesis of cellulose, soluble polysaccharides, and pectins [105, 106]. Studies on oilseed rape seedlings exposed to cold conditions showed significant changes in the content of major polysaccharides [107]. The resistance of the cell walls of the epidermis to frost affects the protection of the protoplast from ice formation, resulting in changes in leaf structure and cell wall thickness in cold conditions [99, 108].

A study of the epidermis ultrastructure in the leaves of Deschampsia antarctica collected during the 20th Ukrainian Antarctic Expedition showed that the morpho-anatomical traits of the leaves collected on the islands Scua and Galindez are similar to those of the species from other regions of the Maritime Antarctic [109, 110, 111, 112]. It was found that all the leaves of the studied species twisted in a tube-shaped curl. The epidermis of leaves of D. antarctica plants collected on the islands Scua and Galindez characterized the next ultrastructural signs. Oval cells of the adaxial epidermis were covered with an even, smooth cuticle layer (Figure 7a and b) [109]. It was shown that leaf blades were downy.

Figure 7.

Ultrastructure of epidermal cells of the leaves of Deschampsia antarctica (a and b) and cytochemical fluorescence of monolignols in D. antarctica leaves (c and d). The syringyl fluoresces in blue; the guajacyl fluoresces in green, and chlorophyll—in red. The histogram in figure e shows the fluorescence intensity of syringyl, guajacyl, and chlorophyll; the abscissa represents the distance (μm) scanned in figure d, which is shown by the white line. Scale bars is 100 μm (a) and = 5 μm (b) [109].

It was early established that low temperatures did not significantly affect the content of lignin, but there were changes in enzyme activity and an increase in the expression of genes coding for C3H protein and cytochrome P450 monooxidase, which are involved in lignin synthesis [113]. An increase in lignin, cellulose, and hemicellulose content was also observed in poplar seedlings and monocots with a decrease in air temperature [114, 115]. The presence of two monolignins (syringyl and guajacyl) in D. antarctica leaf cell walls was revealed (Figure 7c–e) using cytochemical methods and confocal microscopy [109]. The study showed that the content of syringyl and guajacyl in the walls of the adaxial and abaxial epidermis was different, with the highest content of syringyl in the periclinal walls of the upper epidermis and the highest content of guajacyl in the inner walls of both epidermis, the walls of the mesophyll, and conducting bundles. The relative content of syringyl in epidermal cell walls exceeded the relative content of guajacyl by 6–8 times, indicating that the change in lignin components is essential for adaptive changes in response to cold conditions.

Summing up the numerous literature data and the results of our experimental studies, we conclude that regardless of the type of extreme environmental impact on plants, the cells of aboveground organs undergo increased synthesis of lignin. The content and composition of lignin depend on the type of stress and its duration. Cellulose is also synthesized in the cell walls in parallel, regardless of the type of stress. The crystalline form of cellulose, along with monolignins, creates a natural barrier to water transport by inhibiting apoplastic transport, as well as cuticular and stomatal transpiration [30, 115]. It is known that the amorphous zones of cellulose are involved in the transport and absorption of water molecules, while the crystalline form of cellulose does not have this ability [116]. We believe that it is extremely interesting and valuable for fundamental biology and applied sciences to establish the interaction of lignin oligomers with the crystalline form of cellulose-1β [117, 118]. This was experimentally demonstrated using the leaves of Miscanthus giganteus grass. The researchers have shown that the connection between the polysaccharide cellulose and lignin oligomers depends on the molecular weight of the oligomer; the higher the molecular weight of the monolignol, the stronger this connection. Recently, a group of scientists, including López-Albarrán et al. [118], used modeling to study the molecular interactions of oligolignols with cellulose I-β. They concluded that trilignol CA_βO4_SA_ββ_SA and dilignol CA_ ββ_CA were the lignin substructures that most contribute to cellulose adhesion.

In practice, this invention is used in the development of natural wood adhesives. In my opinion, such natural adhesives with lignin oligomers and cellulose can also be used in medicine, textile, and building materials industry. In our opinion, the leaves of reeds and cattails that grow in the coastal strips of European rivers, including the rivers of Ukraine, can serve as a source of such adhesives. Why reeds and cattails? Because the leaves of these plants, in addition to the monolignols (guajacyl and syringyl) and crystalline cellulose, also contain a lot of silicon, which easily absorbs and reflects sunlight. Such properties of natural materials are valuable for their further use in practice.

5. Conclusions

This review indicates that the leaf epidermis plays a role in the adaptation of plants to submergence and drought at structural and biochemical levels. The adaptation mechanisms of the epidermis in leaves to flooding are expressed in the absence of stomata and wax in the leaf epidermis, the loosening of cell walls, and the presence of cuticle pores. Meanwhile, the adaptation mechanisms of the leaf epidermis to drought are expressed in an increase of wax and cuticle, as well as an increase in trichome size and density. The questions involving the epidermal walls of submerged and drought plants are so varied and numerous that this field of plant morphology, anatomy, and cell biology will continue to attract the attention of many investigators to establish not only regularities of fundamental biology but also to use theoretical-experimental data for constructing agricultural plants with resistance to environmental stress.

The adaptation mechanisms of the epidermis of leaves to flooding and drought are also expressed in the synthesis of monolignols syringyl and guajacyl, the content of which depends on environmental conditions. Syringyl and guajacyl monolignols were detected in the leaves of air-water and terrestrial plants using cytochemical methods and laser confocal microscopy. The Pascal program for laser confocal microscopic study of monolignols in the leaf epidermis of hydrophytes revealed that the external environment of the leaf epidermis (water or air) can change the content of monolignols in the epidermis wall. It is established that the anticlinal walls of usual epidermal cells are the main accumulator of guajacyl monolignol. An increase in the content of guajacyl monolignol in the anticlinal walls of the epidermis leads to an increase in buoyancy and the optimization of plant life support on the water surface. It was found that the decrease in soil moisture leads to an increase in the content of monolignols (syringyl and guajacyl, S + G, and ratio of S/G) in the epidermis of plants under drought. An increase in syringyl and guajacyl content in the cell walls of epidermal cells of the leaves of terrestrial plants can reduce cuticular and stomata transpiration and lead to optimization of the water balance of plants grown in moderate drought.

Our experimental data show that the high content of syringyl monolignol, which gives high reed strength in terrestrial plants, can serve as a marker for the use of terrestrial reed plants for making strong bricks for commercial use. The increased content of other guajacyl monolignol in the leaves of air-water plants gives softness to the leaves and can be a marker for getting soft paper from the reeds grown on the banks of the river.

The tolerance mechanism of the leaf to cold is expressed through an increase in syringyl synthesis in the outer wall of the leaf epidermis. Given that syringol monolignol increases the mechanical stability of cells and acts as a barrier to water transport [51] and the action of ice crystals, we can assume that syringol in the periclinal walls of the epidermis of artificial grasses is an active component of plant cold resistance.

Acknowledgments

The author thanks Dr. Klimchuk D.O. for help during scanning electron microscopy of the samples.

Conflict of interest

The authors declare no conflict of interest.

Funding

The preparation and writing of the manuscript chapter (title: “Influence of Environmental Change on Monolignols and the Micromorphology of Leaf Epidermis in Hydrophytes and Terrestrial Plants” for Advances Lignin Technologies) was not funded by M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine.