Role of microstructure and silicon of leaf in adaptation of Quercus robur trees to different light intensity

Role of microstructure and silicon of leaf in adaptation of Quercus robur trees to different light intensity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of microstructure and silicon of leaf in adaptation of Quercus robur trees to different light intensity Оlena Nedukha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8995237/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The microstructure, localization, and content of silicon inclusions in the leaf epidermis of Quercus robur trees grown in forest-steppe zones of southern Ukraine with different sunlight intensities were studied with the electron microscopic method and laser confocal microscopy. We established the influence of sun intensity on silicon content in oak leaf epidermis. It was found that silicon is most accumulated in trichomes, stomata, and ordinary epidermal cells of oak leaves. The observed phenotypic plasticity of leaves is manifested in an increase in the trichome number and stomatal density, as well as in a decrease in leaf size and area in trees growing in direct sunlight. Common oaks from the southern region of Ukraine are able to grow normally and adapt to high levels of solar radiation due to their increased ability to absorb and reflect light by leaf surface. The various characteristics of these leaves can be used for practical applications. Leaf micromorphology Quercus robur Epidermis structure Light intensity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Studying the structural and functional organization of Quercus robur Leaves are crucial for botany and forestry in Ukraine and Europe, as they are a major species forming forests. The practical utilization of oak leaves, bark, seeds, and acorns in phytochemistry and pharmacology adds significant value to this research from both fundamental and applied perspectives. Phenolic acids, terpenoids, and tannins derived from oak exhibit anti-inflammatory, anti-diabetic, and antitumor properties. These compounds hold significant promise as potential drug candidates in the fight against infectious diseases (Taib et al. 2020 ). The conservation of oak forests in Ukraine is a growing concern, as climate change brings about more droughts, alongside the impact of atmospheric and soil pollution caused by the war with Russia, which has been ongoing since 2022. Such factors adversely affect the sustainability of oak forests. (Osborn and Taylor 1990 ; Novak 2005 ). Conserving the common oak is a crucial measure for preserving Europe's and Ukraine's natural landscapes. Additionally, oak stands minimize soil erosion by decreasing sediment Sancho-Napik and enriching the surrounding soil with nutrients (Broome et al. 2021 ). Tree leaves are often the first to detect environmental changes and respond appropriately. It is known that the morphological traits of oak leaves are adapted to the microclimate of their growth, as demonstrated in previous work on leaf anatomy (Sancho-Knapik et al. 2021 ; Alonso-Forn et al. 2023 ). A wide sample of oak species from around the world has been shown to develop a set of leaf morphological traits with different ecological roles (Martín-Sánchez et al. 2024 ). The outer cell walls of the leaf epidermis serve primarily as both a barrier for penetration and a primary transport route for sunlight, CO₂, and water, while also acting as the interface between plant organs and the environment (Riederer and Schreiber 2001 ; Bacelar et al. 2004 ; Müller and Riedere 2005 ; Lewandowska et al. 2020). Epidermal walls are where silicon inclusions are synthesized (Wang et al. 2005 ) and are crucial in regulating sunlight absorption by the surface of the leaf (Mirshafieyan and Gue 2014). The functioning and structure of leaf epidermis rely on temperature, humidity, and UV intensity. The steppes of Ukraine experience temperatures ranging between + 22 and + 30°C and an average annual rainfall of 400–600 mm. Trees growing in this area are adapted to severe drought. Don’t overlook the fact that they are extensively researched; there are several studies, but natural and human-caused disasters in oak forests are researched extensively. However, there is a lack of understanding about important issues such as the phenotype plasticity of the species and the regeneration of oak stands in the steppe zone of Ukraine. This knowledge serves as the foundation for the conservation of oak forests' biological diversity. We assume that the growth of oak trees in southern Ukraine, where conditions feature strong solar radiation and limited water availability, may be attributable to specific traits of leaf epidermal structure withstanding the region's hot climate. In addition, it is known that under conditions of heat, plants synthesize wax in epidermal cells (Reynhardt and Riederer 1994 ; Kerstiens 1996 ; Rahman et al. 2021 ), as well as amorphous and crystalline silicon (Hiroyuki Takeda et al. 2013 ; Grašič et al. 2020 ), which can reflect and absorb sunlight, and thus these two components of the epidermal walls can inhibit transpiration processes in leaves, optimizing the water balance of the plant. Therefore, the aim of this study was to investigate the role of the structural-functional signs of the oak leaf epidermis in the adaptation of the species to high solar irradiation in the southern forest-steppe of Ukraine. In addition, the second goal was to compare the role of leaf wax and silicon inclusions in the adaptation of Quercus robur trees to growth in conditions of strong shading and direct sunlight. Material and methods Plant Material (Fig. 1 ) and Environmental metrics Leaves of common oak, Quercus robur L. Fagaceae) were collected in june from trees grown in the forest-steppe zone of the Dnepropetrovsk region of Ukraine (48°27′58″N, 35°01′31″E, 139 m above sea level). The sampling site of Q. robur has the following habitat characteristics: 1: A forest of the southern ravine-oak variety grows on the edge of a stand near the village of Bashmachka in the Solonyansky district of the Dnepropetrovsk region, on the right bank of the Dnipro River in Ukraine. At site 2, on the top of a ravine, two oaks (Nos. 1 and 2) grow, while site 1 hosts oaks (Nos. 3 and 4). Refer to Table 1 for more information. Specifically, Oak No. 1 grows at the forest edge, Oak No. 2 grows in dense forest, and Oak Nos. 3 and 4 grow alone. In late June, Ukraine's Dnepropetrovsk Region had hot weather. The average daily air temperature in June was significantly higher than normal. For 3–6 days, the maximum air temperature surpassed 30°C and reached 33–37°C on the hottest days. The average 10-day air temperature was 1.2–3.8°C above the multiyear average and ranged from + 20.6–23.0° C (source: https://www.apk-inform.com/uk/crop/1527945 ). There was a precipitation deficit for most days during the ten-day period. High air temperatures and a significant lack of precipitation led to intense moisture loss, particularly in the upper soil layer. The region experienced an ongoing crop drought (source: https://www.apk-inform.com ). On the day of collection, the weather was sunny. The amount of light on the leaf surface differed between oak trees #1, #2, #3, and #4. PPFD (photosynthetic photon flux density) levels on the adaxial surface of oak #1 leaves were 900 µmol·m -2 ·s -1 ; for oak #2, it was 50 µmol·m⁻²·s⁻¹; for oak #3 and oak #4, the PPFD was 1500 µmol·m⁻²·s⁻¹. The LI-250 light meter (USA, LI-COR) was used to measure the PPFD. Light intensity was measured on the upper surface of the lowermost tier of leaf plates for cytological analyses. 15–20 leaves were collected from each tree's branches, and their length and breadth were measured. To carry out these analyses, we used the centre portion of every other (upper) lobe from five leaves of each type (Fig. 1 ). The leaf area was determined following the procedure outlined by Pandey and Singh ( 2011 ). Each leaf was placed on a sheet of graph paper with millimetre markings, and the outline was traced. The sheet area was then measured. A section of graph paper, equivalent to the leaf's outline, was cut using a paper knife and weighed on an electronic scale. Similarly, one square centimetre of the same graph paper was cut and weighed. Leaves from the same sample were measured multiple times. The area of the leaf was calculated using the non-destructive method with the following equation: Leaf area can be determined using the following formula: Area (square cm) = weight of paper within the outline (g) / weight of paper within the outline (g). Scanning electron microscopy The middle portion of each second upper lobe of the leaf plate was fixed in a solution consisting of 2% paraformaldehyde and 1% glutaraldehyde (1:1, vol.) in 0.5 M phosphate buffer (pH 7.2) for 13 hours at +4 o C. After bringing them to the laboratory, wash the samples with the same buffer and dehydrate them in a series of alcohols (70%, 80%, and 100% ethanol; twice every 30 minutes) as per Talbot and White's (2013) protocol. After dehydrating the samples, they were mounted on on stubs, coated with carbon and gold, and analysed using a JSM 6060 LA scanning electron microscope at 30 kV. Cell size was determined by measuring 30–40 usual epidermal cells, 30–35 stomata, and 20–25 trichomes in three samples from each of the five leaves. All measured variables were then analysed for variance using the Origin 6.1 program. Laser confocal microscopy Samples for laser confocal microscopy were prepared following Dabney et al.'s ( 2016 ) procedure. We exposed sections from the middle of the second (upper) leaf blade of the leaf plate of five leaves from each oak tree (10 × 20 mm) to + 220°C for three hours until the samples turned grey. The study analysed the presence of silicon in leaf samples employing an LSM5 laser scanning confocal microscope (Zeiss, Germany) with excitation and emission wavelengths of 480 nm and 530 nm, respectively. The study considered five leaves from each tree. The silicon fluorescence intensity was then evaluated using the Pascal program on the LSM5. Statistics Statistical differences among mean values were determined using the Student’s t-test with a significance level of P < 0.05. Significant differences were observed across all measurements. Origin 6.1 software was employed for data analysis. Each experiment was conducted three times. We measure leaf size, trichome and epidermal cell size, stomata density, and the microstructure of the leaf surface, or the cytochemical study of the fluorescence of silicon inclusions. For cytological research, we chose 20–22 medium-sized leaves from the lower branches of every oak tree. Ten leaves from each variant were chosen to measure linear dimensions, and five leaves were selected for studying the microstructure of the leaf epidermis and five leaves for conducting cytochemical research on silicon. The average size of the leaf blades for the four oak trees is shown in Table 2 . The length of the blade, from the apex to the petiole, and the width at the level of the second upper lobe are the first and second values, respectively. The mean value comparison between leaves was done through ANOVA. Origin 6.1 programs performed the analysis of variance for all measured variables. Results Micromorphology of Quercus robur L. leaves ( Fig. 1 – 5 ) The surface structure of the leaves of four oak trees was studied. Trees No. 1 and No. 2 grew in the shade in a group of trees on the edge of the forest near the village of Bashmachka in the Dnipropetrovsk Region. Trees No. 3 and No. 4 grew individually on ravine tops in direct sunlight without shade (see Table 1 for the basic parameters of the samples). Leaves from four selected common oak ( Q. robur ) trees, regardless of age or growth location, display comparable leaf blade characteristics: the leaves have a short petiole, are hypostomatic (stomata present only on the lower surface of the blade), are elongated-obovate in shape (as shown in Fig. 1 ), are narrowed downwards, and are pinnately lobed. We found that the leaf blades are rounded and blunt, with shallow notches between them. Some variations in leaf size, area, and ultrastructure were observed in the leaf epidermis. Table 2 displays the average leaf blade size of the four trees studied. Scanning electron microscopy (Fig. 2 – 5 ) Leaves of shaded trees. Tree No. 1. Adaxial surface . The oak leaves under study exhibited an irregular upper epidermal surface. The periclinal walls of epidermal cells protruded above the surface. Such walls form spherical or ovoid bulges, around which anticlinal walls produce depressions that cover needle-like and lamellar waxy structures, varying in size from 1 to 4 µm (Fig. 2 a, b; Table 3 ). Stomata and trichomes were absent from the upper epidermis. The periclinal wall protrusions exhibit a partially smooth surface, sometimes coated with wax-like structures, reminiscent of the appearance of the anticlinal recessed walls. Figure 2 Scanning electron microscopy of adaxial ( a, b ) and abaxial ( c, d, e, f ) leaf surfaces of Quercus robur tree No. 1, which was grwn in the shaded zone of Dnipropetrovsk Region, Ukraine. The yellow arrows indicate the location of wax-like crystals. Abbreviations used include A.w - anticlinal wall, P.w – periclinal wall, St – stomata, Tr – trichrome, and W – wax-like structure The abaxial epidermis . The lower epidermis of oak leaves contains stomata and trichomes, which originate from the lower epidermal cells and the cells of the protruding veins (Fig. 2 c, 2 d, 2 e, and 2 f). We established that the tested hypothesis regarding the differences in ultrastructural characteristics of leaves including stomata and trichome density in shaded trees and trees under direct sunlight was true. The lower epidermal cells are covered by a layer of lamellar crystalline structures, making it difficult to see and measure their boundaries and sizes. Table 3 provides details on the density of stomata and trichomes, as well as the average size of stomatal cells and trichomes. The stomata are oval-elongated, with guard cells covered by thin crystalline structures approximately 2 µm in size. While the stomatal gap is free of crystalline structures. Trichomes on the leaf surface are simple, slightly raised, and unbranched, with a drop-shaped base; rounded-plate shaped main parts, and pointed speices. The base of the trichomes is covered by small, stick-shaped crystalline structures, sometimes accompanied by larger, crystal-like structure Trichomes on the leaf surface are simple, slightly raised, and unbranched, with a drop-shaped base, rounded-plate-shaped main parts, and pointed species. The base of the trichomes is covered by small, stick-shaped crystalline structures, sometimes accompanied by larger, crystal-like structures (see Fig. 2 f). Tree No. 2. Adaxial surface. The upper surface of leaves from tree number 2 is distinct, with clearly defined epidermal cell contours and an absence of stomata and trichomes (see Fig. 3 a, b). Anticlinal walls protrude above the epidermal surface, forming a tall fence around each cell. The cells on the upper leaf surface are small and have a similar shape to those on tree number 1 (refer to Table 3 ). The periclinal walls are depressed, and both the anticlinal and periclinal cells are uniformly covered with crystalline structures measuring 1 to 3 µm in size. On the abaxial surface , the epidermal cells are concealed by a continuous layer of lamellar crystalline structures, while stomata and trichomes are present (see Fig. 3 c, d, e, f). The size of the main cells cannot be determined, as seen in sample 1. Additionally, fine crystalline structures that measure approximately 2 µm enclose the stomatal guard cells. The mean density of trichomes is 71 ± 7.0 per square millimeter of surface area. Trichomes extend from the cells of protruding veins and are positioned above the usual cells. They are simple, unbranched, and slightly raised, with a distal base that is drop-shaped and a middle part that is rod-shaped and pointed at the apex. The trichome base is covered with small, crystalline, rod-shaped structures. The average sizes of stomatal guard cells and trichomes are presented in Table 3 . Leaves of unshaded trees in sun zone. Tree No. 3. Adaxial Surface : An analysis of the leaf ultrastructure of Tree #3 revealed that periclinal walls of the epidermal cells protruded above the surface, creating spherical or ovoid bulges where the anticlinal walls formed depressions. These depressions were covered in needle-like and lamellar waxy structures ranging in size from 1 to 4 µm (refer to Fig. 4 a, b). Stomata and trichomes were absent on the upper epidermis. The surface of the periclinal wall protrusions is partially smooth, sometimes covered with waxy structures, similar to the surface of the anticlinal recessed walls. The cell sizes on the adaxial surface are listed in Table 3 . Abaxial Surface : On the lower surface of the specimen leaf, stomata and trichomes are visible (refer to Fig. 4 c, d, e, f). Trichomes emerge from the pavement cells and from the cells of protruding veins. The stomata are slightly elongated and oval-shaped. The density and size of stomata are listed in Table 3 . Stomatal guard cells are covered with thin crystalline structures measuring about 2 µm, while the stomatal gap is free of such structures. The average size of the stomatal gap measures 18 ± 0.6 x 5 ± 0.2 µm. The stomatal surrounding pavement cell surface is also coated in a continuous layer of lamellar crystalline structures, which renders the boundaries of the ordinary cells of the lower epidermis indiscernible. The trichomes are situated above the primary epidermal cells, tilted slightly upward, and occasionally above the veins. The apex base of the trichomes assumes a drop shape; the main part is plate-shaped and crystalline. The lower epidermal usual cells surrounding the stomata and the cells beneath the trichomes are covered by a continuous layer of lamellar crystalline structures. As a result, the borders of the usual cells surrounding the stomata are not visible, which is similar to the structure of the epidermal cells in the leaves of trees No. 1 and No. 2. The trichome base is also covered with small, rod-shaped crystalline structures and occasionally has larger crystal-like structures as well (refer to Fig. 4 f). The average sizes of stomatal closure cells and trichomes are provided in Table 3 . Leaves of tree No. 4. Adaxial Surface The ultrastructure analysis of the adaxial leaf surface of Tree No. 4 revealed the absence of trichomes and stomata. The epidermis surface was composed of typical epidermal cells, with periclinal walls projecting above the surface and anticlinal walls recessed (Fig. 5 a, b). The spherical or ovoid bulges protruding from the periclinal walls of the epidermal cells resulted in depressions formed by the anticlinal walls. These depressions were covered in needle-like and lamellar wax structures of varying sizes, ranging from one to four µm. Both the anticlinal walls and parts of the periclinal walls were covered in waxy structures. Stomata and trichomes were not present on the upper epidermis. The periclinal wall projections of most cells have a smooth surface, although some periclinal walls and the surface of the anticlinal recessed walls are occasionally covered with waxy structures. Table 3 presents the cell size on the adaxial surface. Abaxial Surface The leaves of Oak No. 4 exhibit stomata and trichomes (Fig. 5 c, d, e, f) that originate from the main epidermal cells and the vein cells. The stomatal guard cells are coated with needle-like waxy structures that measure approximately 2 µm. The stomatal gap lacks crystalline structures with an average size of 17 ± 1.3 x 5 ± 0.2 µm. The lower epidermal cell surface that encircles the stomata features a constant waxy layer in diverse forms—needle-like,lamellar, and crystalline. Trichomes sit atop the main epidermal cells, being slightly lifted and occasionally positioned over the veins. They are simple, raised, and unbranched with an elongated or oval base and a rod-shaped head. The trichome base is encompassed with needle-like waxy structures. The boundaries of the usual cells in the lower epidermis are rendered invisible by the continuous layer of lamellar crystalline structures that cover the surface of the epidermal cells surrounding the stomata, a feature consistent with previous specimens. The average size of stomata cells and trichomes can be found in Table 3 . Laser Confocal Microscopy of Silicon in Quercus robur leaf epidermis ( Fig. 6 , 7 ) Leaves of tree No. 1 grown in shade zone. Our study shows the bright green fluorescence of silicon inclusions in the epidermal cells of oak tree No. 1 leaves (Fig. 6 a, b, 6 d). The fluorescence of the silicon was detected in the anticlinal and periclinal walls of the adaxial epidermis (Fig. 6 a). The silicon inclusions were densely arranged in the anticlinal walls, forming nearly continuous layers. Individual silicon inclusions are visible in the periclinal walls. Silicon fluorescence is noticeable in the walls of usual epidermal cells, guard cells of stomata, and at the base of trichomes in the lower epidermis. The silicon had a single, individual inclusion in its form (Fig. 6 b). The intensity of the silicon fluorescence profile varied among the studied cells and depended on the leaf surface and cell type (Table 4 ; Fig. 6 c, c', d, d'). The silicon intensity varied between the anticlinal and periclinal walls of epidermal cells on the adaxial surface. The intensity in anticlinal walls was 120 ± 13 relative units, while in periclinal walls, it was significantly lower, quantifying to 70 ± 5.3 relative units. The intensity of silicon in the anticlinal walls of ordinary cells of the lower epidermis was 75 ± 8.8 relative units. In contrast, in the periclinal walls of those cells, it was 70 ± 5.3, and in trichomes and the closing cells of stomata, it was 156 ± 11 and 71 ± 5.4 relative units, respectively. The silicon inclusions are most densely concentrated in the anticlinal walls of the adaxial epidermis and in the trichomes of the abaxial epidermis. Leaves of tree No. 2 grown in shade zone. The analysis showed green silicon fluorescence in the cell walls of epidermal cells in both the upper and lower epidermis, as well as in stomata and at the base of trichomes in Q. robur leaves of tree No. 2, which were grown in shade (Fig. 6 e, f). Additionally, silicon fluorescence was detected in the anticlinal and periclinal walls of the main epidermal cells of the adaxial epidermis (Fig. 6 g). The relative units of the fluorescence intensity were 105 ± 11.0 and 55 ± 4.3, respectively. Silicon fluorescence was also found in the guard cells of stomata, at the base of trichomes, and in the anticlinal and periclinal walls of epidermal cells in the abaxial epidermis, consistent with the previous sample. The intensity of the silicon profile varied depending on the leaf surface and cell type, as shown in Table 4 and Fig. 6 g, g', h, h'. Silicon levels were 80 ± 9.3 in anticlinal walls, 23 ± 1.7 in periclinal walls, 121 ± 12 in trichomes, and 65 ± 7.1 in stomata, all reported in relative units. In summary, the periclinal walls of the primary cells on the upper and lower surfaces, including the walls of stomata, have low profile intensity. Leaves of tree No. 3 grown in sunnier area. A cytochemical analysis of silicon in Q. robur leaves was conducted on tree No. 3, which grew in direct sunlight without shade. The results revealed green fluorescence of silicon in the walls of upper epidermal cells, trichomes, stomata, and conducting bundle cells (Fig. 7 a, b, g, h). Silicon fluorescence was also observed in various parts of the leaf, including the abaxial epidermis cells, guard cells of stomata, and trichome base. The intensity of the silicon profile varied depending on the leaf surface and cell type, measured in relative units. The highest intensity was found in the anticlinal and periclinal walls of the upper epidermis, with values of 140 ± 12 and 124 ± 11 relative units, respectively. Similarly, the intensity of the profile was high in the anticlinal walls of the lower epidermis, at the base of trichomes and stomata guard cells, with values of 100 ± 9.7, 180 ± 15, and 125 ± 12 relative units, respectively. However, the periclinal walls of lower epidermal cells showed a lower profile intensity of 80 ± 5.4 relative units (See Table 4 ; Fig. 7 c, c', d, d'). Leaves of tree No. 4 grown in sunnier area.. The cytochemical analysis revealed green fluorescence for silicon within the walls of ordinary epidermal cells, trichomes, stomata, and conducting bundle cells. In the adaxial epidermis, silicon fluorescence was detected in the anticlinal and periclinal walls of the upper epidermal cells, similar to the previous sample (see Fig. 7 e, f). Silicon fluorescence was also detected in the anticlinal and periclinal walls of the abaxial epidermal cells, guard cells of stomata, and trichome bases. The intensity of the silicon profile varied based on the leaf surface and cell type, as indicated in Table 4 and Fig. 7 g, g', h, h'. The highest silicon profile intensity was in the anticlinal and periclinal walls of the upper epidermis, with relative units of 145 ± 13 and 115 ± 12, respectively. Additionally, significant profile intensity was found in the anticlinal walls of the lower epidermis, trichomes, and stomatal guard cells, with values of 120 ± 11, 185 ± 12, and 128 ± 11 relative units, respectively. Conversely, the periclinal walls of ordinary cells in the lower epidermis demonstrated lower profile intensities, measuring just 95 ± 7.7 relative units. Discussion We have studied the leaves of Q. robur trees growing in different conditions in the forest-steppe zone (both in the forest and on the slopes of ravines) of the Dnepropetrovs'k region of Ukraine. We found similarities and differences in leaf blade size, leaf surface ultrastructure, localization, and content of silicon inclusions in the studied tree leaves depending on the light intensity of the environment where the trees grewn in sunnier or shade zones. Furthermore, we found that the tested hypothesis about the differences in leaf size between shaded trees and trees in direct sunlight was correct. The leaf blades of the first two trees, located on the forest edge and shaded by other trees (trees No. 1 and No. 2), were significantly larger than those of trees No. 3 and No. 4 growing without shade on ravines. The leaf, being the most adaptable organ to environmental changes (Nevo et al. 2000 ), shows the effects of environmental factors more clearly than stems and roots. Smaller leaf sizes can result in reduced water loss, especially from the adaxial surface. Such a phenomenon has been observed in the leaves of two-year-old saplings of Quercus petraea and Q. robur , as reported by Dupouey and Dreyer ( 2004 ), and in Olea europaea trees growing in environments with high air temperatures and low water supplies, as found by Bacelar et al. ( 2004 ). Plants typically respond to shade by producing larger leaves with a lower mass per unit area, allowing for more efficient light capture per unit mass, as noted by Niinemets and Sack ( 2006 ). Our results of studying the size of oak leaves showed that oak trees growing in direct sunlight are characterized by small leaves. Our data are confirmed by other researchers. It is well-established that trees growing in sunnier areas produce smaller leaves with greater mechanical strength (Onoda et al. 2008 ). Additionally, these leaves have thicker cuticles and epicuticular wax layers. This phenomenon is also observed in the leaves of succulent and mesophytic plants (Richardson and Berlyn 2002 ; Lewandowska et al. 2020). The unshaded leaves of plants that grow in direct sunlight are characterized by an increase in vessel and stomata density compared to shaded leaves (Karabourniotis and Bornman, 1999 ; Karabourniotis et al. 2020 ). Granier and Tardieu ( 1999 ) determined that the size of unshaded leaves is significantly affected by light intensity, which can impede cell division and elongation. These authors have shown that the rate of cell division and rate of cell elongation decreased with increased illumination. This phenomenon can be attributed to shading as a prevailing abiotic stressor, given its occurrence as a result of light obstruction by neighboring plants. Our research indicated that shaded oak trees (No. 1 and No. 2) have fewer simple non-glandular trichomes in their leaves compared to oak trees growing in non-shaded conditions (No. 3 and No. 4). The increase in trichome density in the unshaded oak trees (No. 3 and No. 4) is likely due to their role in protecting the leaves from environmental stressors. Simple elongated trichomes have been found in the leaves of various tree and grass species, including Q. laevis , red oak ( Q. rubra ) (Kryvoruchko and Bessonova 2018 ), and black walnut ( Juglans nigra ) (Prasad and Gülz 1990 ; Wagner et al. 2004 ; Chen et al. 2007 ; LoPresti 2015 ; Oksanen 2018 ). The increase in trichome density in unshaded oaks is thought to be related to their function as a barrier against UV radiation, increased transpiration, and pathogens (Wang et al. 2021 ). These structures are crucial for the development of organs and are supported by phenolic compounds, specifically flavonoids. The formation of trichomes and the accumulation of phenolic compounds are linked at the molecular level. As non-glandular trichomes develop, they show significant morphological similarities to glandular trichomes. As they continue to grow and the secondary wall thickens, phenolic substances are moved to the cell walls of the trichomes (Chen et al. 2007 ; LoPresti 2015 ). Trichomes act as optical filters, protecting sensitive tissues from UV radiation by screening out harmful wavelengths through the scattered deposition of phenolics in the cell walls. The increased light reflectance on the surface provides protection against strong visible radiation (Skaltsa et al. 1994 ; Karabourniotis et al. 2020 ). Our research, in conjunction with the aforementioned publications, led us to conclude that the leaf trichomes of oaks No. 3 and No. 4 protect the epidermis from high solar irradiation and reduce the intensity of cuticular transpiration. We observed that the intensity of illumination did not affect the presence of wax-like inclusions on the cell walls of the epidermis, the closing cells of stomata, and the base of trichome on the adaxial and abaxial surfaces of leaves from four Q. robur trees in Ukraine's forest-steppe zone. Wax can exist in cells in two forms: bound to the cuticle or free, creating crystals of different shapes on the cell surface (Kerstiens 1996 ). The wax serves as a crucial barrier against unregulated water loss in leaves, has the capability to absorb or reflect UV radiation, and suppresses cuticular transpiration (Riederer and Schreiber 2001 ). The presence of waxes has been noted in the leaves of different tree species (Prasad and Gülz 1990 ; Prasad et al. 1990 ). The simultaneous presence of crystalline, liquid, and amorphous wax phases could be distinctive characteristics of various organs of numerous plant species (Reynhardt and Riederer 1994 ; Müller and Riederer 2005). The permeability of the amorphous phase is determined by the degree of crystallinity of the wax, which affects the effectiveness of the diffusion barrier. The study concludes that epicuticular waxes remain on the leaf surface and limit access to the pores in the cuticle. Based on the data regarding the role of wax on leaf surfaces and our experiment results on the existence of wax-like structures on the anticlinal walls of the upper epidermis and on all types of cells of the abaxial epidermis of leaves from four Q. robur trees, we can deduce the following suggestion: Crystalline wax-like structure is a structural hallmark of oak leaves, regardless of the light intensity, and it regulates transpiration and optimizes the leaf blade's water balance. We revealed the presence of silicon in leaves of investigated oak trees. It is known that silicon in plant cells functions as a biological lens, helping incident infrared light penetrate efficiently inside cells and leaves when combined with carbohydrates, proteins, or lipids in the cell walls (Guerriero et al., 2016; Grašič et al., 2020). These properties can facilitate efficient heating of plants as absorbed far-infrared light is converted into heat (Ma et al., 2011; Hiroyuki Takeda et al., 2013). Silicon inclusions (8–12 µm in size) in the epidermis effectively cool leaves via highly efficient thermal radiation in the mid-infrared range, according to Wang et al. ( 2005 ). Stomatal transpiration also cools leaves. Reduction of transpiration in plants can be achieved by depositing silica structures in the leaf epidermis. This approach may be practical and environmentally friendly for improving plant resistance to high temperatures. The results indicate that plants infused with biosilicon and subjected to temperature stress employ an uncomplicated physical strategy to defend against heat stress. Silicon is capable of absorbing light fluxes ranging from infrared to ultraviolet. In plants, the refractive index of silicon structures is approximately equal to the wavelength of visible light. As a result, not only do such structures absorb light, but they also reflect it in the region of wavelengths referred to as the photonic band gap (Skaltsa et al. 1994 ; Large 1998 ; Yahaya et al. 2013 ). This optical phenomenon is due to the slowing down of light's group speed at the edges of the photon zone. In this scenario, the interaction between light and materials is intensified. Periodic silicon structures facilitate the enhancement of light reflection and absorption, while higher plants possess characteristic structures that sense infrared or far-infrared radiation. Pedrotti and Leno ( 2007 ) indicate that the radius of curvature and size of an object are significant parameters for light reflection. Sunbeams can remain unaltered in direction based on the thickness and curvature of the lens. The periclinal walls of the upper epidermis of oak leaves, which we examined, exhibited diverse shapes, either convex or concave depending on the degree of illumination. We were able to identify them as a convex lens (oaks No. 1, 3, and 4), a concave lens (oak No. 2), or a cylindrical lens, which corresponds to the anticlinal walls of the epidermis of all examined samples. The question of UV ray transmission, absorption, and reflection intensity through cell walls acting as lenses remains unanswered and requires further study by biophysicists. Researchers recently discovered silicon-rich trichomes on both the upper and lower leaf surfaces of the Aphananthe aspera (Muku tree) in Osaka, Japan (Hiroyuki Takeda et al. 2013 ). The team used infrared spectroscopy to analyze the trichomes and found that they play a significant role in absorbing far-infrared light due to specific chemical bonds in organic materials and silica. Our research in southern Ukraine confirmed the presence of silicon inclusions in the upper and lower leaf epidermal cells of all oak trees surveyed. The magnitude of the silicon profile varied depending on the type of epidermis and cell. Observations showed that oak trees grown in direct sunlight had greater silicon fluorescence in the cell walls of trichomes, stomata, and upper and lower epidermal cells. This led to the conclusion that increasing silicon content in these areas enhances sunlight absorption and reflection, optimizing photosynthesis and thermal stability. Our data show that the use of leaf blades of oaks growing under direct solar irradiation is promising for the use of biological forms of silicon, which has potential applications in medicine, pharmacology, and agriculture. Plants with high silicon content, such as oak trees, have been found to be beneficial for bone tissue restoration in medicine (Taib et al. 2020 ). Additionally, the use of oak leaves as fertilizer has shown positive results in agriculture, particularly in improving plant health during water stress conditions. The application of oak leaves as fertilizer in agriculture yields positive results by optimizing the researchers found that adding oak leaf powder and biofertilizer to soil improved the growth and biochemical composition of tomato leaves of several genotypes under water stress conditions (Nawroz Abdul-Rezzak et al. 2022). In conclusion, this study highlights the potential benefits of oak leaves for mitigating water stress and improving plant life, making them promising for practical applications. Conclusions The study of the leaves of Quercus robur trees grown in sunnier areas or shade zones in the southern forest-steppe zones of Ukraine revealed phenotypical plasticity and adaptive structural signs of oak leaf blades to changes in sunlight lighting. These differences, such as leaf size, density of trichomes and stomata, and a bulge in the periclinal cell walls in leaf epidermis, may reflect adaptations to the intensity of sunlight. These features help reflect sunlight from the upper surface of sunlit oak leaves compared to shaded leaves. The presence of such structural features shows that oak leaves are characterized by the ability to adapt to high solar irradiation and reserve optimal water balances in the leaves. The presence of such structural features shows that oak leaves are characterized by the ability to adapt to high solar irradiation and reserve optimal water balances in the leaves. A laser confocal microscopic study of silicon inclusions present in the leaf epidermis of Q. robur revealed that the external environment—sunnier areas or shade zones—can change the content of silicon in the leaf epidermal wall. Leaves of trees grown in strong solar irradiation contained significantly higher values of silicon inclusions in cell epidermis. We assume that an increase in the silicon content in the epidermis of oak leaves growing without shade leads to a reduction in cuticular and stomatal transpiration and optimization of the water balance of oaks growing in direct sunlight, which helps trees adapt to the climate and aridization under the action of strong solar irradiation. epidermis, Declarations Acknowledgements The author is grateful to Dr. Shevchenko G. for help during collecting samples for the study, and Dr. Topchiy N. for help during measure the sun intensity on the tree leaves' surface. Funding . The study was financially supported by the National Academy of Science of Ukraine; the studies were performed in the Department of Cell Biology and Anatomy of Institute of Botany of National Academy Sciences of Ukraine; № 8-22, 2022-2023 ,state registration number; title of project - Determination of structural-functional and molecular features of resistance of common oak ( Quercus robur L.) to aridization of the Ukrainian climate (2022-2023). Compliance with ethical standards Consent for publication The manuscript is not published elsewhere. Conflict of interest The author declares that she has no conflict of interest. Author contributions Dr Nedukha Olena designed the re­search methodology, fixed and photographed samples, examined those using electron and laser confocal microscopy, described the results, and wrote a manuscript of the article collected and analyzed the data, and drafted the manuscript. 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Supplementary Files 4TablesNedukha1.docx Supplemantarymaterials.doc Listofpossiblereviewers.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8995237","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607645472,"identity":"498defcf-9264-4af8-a1af-bf8f33d223b3","order_by":0,"name":"Оlena Nedukha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYLACHoaEBAb2Hgibj3gtPGcYGA4A2WzEa5HIAWthIKiFn4H34IO3bWl5/DPfHnz8McdOho2B+eGjG3i0SDbwJRvObcsplridl2xwcFsy0GFsxsY5eLQYHOAxk+Ztq0hsuJ1jJnFwGzNQCw+bNAEt5r9BWubfPAPSUk+UFjNm3racxA03eEBaDhPWItnMYyw551xa4sYzOcYGZ7cd52FjJuAXfvYeww9vypIT5x0/Y/igclu1PT9788PH+LQwMBMhMgpGwSgYBaOAVAAAerNFOrF1dyUAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Botany of National Academy of Sciences of Ukraine","correspondingAuthor":true,"prefix":"","firstName":"Оlena","middleName":"","lastName":"Nedukha","suffix":""}],"badges":[],"createdAt":"2026-02-28 12:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8995237/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8995237/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104972104,"identity":"690c6b3b-7a21-46a1-a9f4-03b07a91b680","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107261,"visible":true,"origin":"","legend":"\u003cp\u003eCommon view of leaf blades from four \u003cem\u003eQuercus robur \u003c/em\u003etrees was grown in Dnipropetrovsk Region, Ukraine. Trees No. 1 and No.2 was grown among other trees in the shade zone; trees No. 3 and No. 4 was grow in direct sunlight in sun zone. Bars = 20 mm\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/941e80e2c3a9875946760c09.jpeg"},{"id":104972111,"identity":"d87d4bc2-c001-4728-95b4-65beef819158","added_by":"auto","created_at":"2026-03-19 11:15:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":440327,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy of adaxial (\u003cstrong\u003ea, b\u003c/strong\u003e) and abaxial (\u003cstrong\u003ec, d, e, f\u003c/strong\u003e) leaf surfaces of \u003cem\u003eQuercus robur \u003c/em\u003etree No. 1, which was grwn in the shaded zone of Dnipropetrovsk Region, Ukraine. The yellow arrows indicate the location of wax-like crystals. Abbreviations used include A.w - anticlinal wall, P.w – periclinal wall, St – stomata, Tr – trichrome, and W – wax-like structure\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/9d001d5ca2ff0adaa38bf445.jpeg"},{"id":104972110,"identity":"8a0a15f4-2693-4056-b319-8cf4ec38680e","added_by":"auto","created_at":"2026-03-19 11:15:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":445485,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy images of adaxial (\u003cstrong\u003ea, b\u003c/strong\u003e) and abaxial \u003cstrong\u003e(c, d, e, f\u003c/strong\u003e) surfaces leaves from \u003cem\u003eQuercus robur \u003c/em\u003etree No. 2\u003cstrong\u003e,\u003c/strong\u003e was grown in the shaded zone of Dnipropetrovsk Region in Ukraine. \u0026nbsp;Abbreviation: A.w – anticlinal wall, P.w – periclinal wall, St- stomata, Tr – trichrome, W – wax-like structure\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/bde90874253e3e04131a6949.jpeg"},{"id":104972107,"identity":"38bd3fe2-3433-46c0-9e0b-085d82c66935","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":459975,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy of adaxial (\u003cstrong\u003ea, b\u003c/strong\u003e) and abaxial (\u003cstrong\u003ec, d, e, f\u003c/strong\u003e) leaf surfaces of \u003cem\u003eQuercus robur \u003c/em\u003etree\u003cstrong\u003e \u003c/strong\u003eNo. 3 was grown in sun zone of forest of Dnipropetrovsk Region, Ukraine. \u0026nbsp;A yellow arrow indicates wax-like crystal (fig. \u003cstrong\u003ef\u003c/strong\u003e). Abbreviation used: A.w – anticlinal wall, P.w – periclinal wall, St – stomata, Tr – trichrome, W –- wax-like structure\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/5a958b6e23b051aa9774ee26.jpeg"},{"id":105035218,"identity":"ef29c10d-e244-4585-8cea-e399882ad806","added_by":"auto","created_at":"2026-03-20 07:25:40","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":431298,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy images of the adaxial (\u003cstrong\u003ea, b\u003c/strong\u003e) and abaxial (\u003cstrong\u003ec, d, e, f\u003c/strong\u003e) leaf surfaces of \u003cem\u003eQuercus robur \u003c/em\u003etree\u003cstrong\u003e \u003c/strong\u003eNo. 4, which was grown in \u0026nbsp;sunnier area of\u003cstrong\u003e \u003c/strong\u003ethe forest of Dnipropetrovsk Region, Ukraine. The following abbreviations are used: \u003cstrong\u003eFig. \u0026nbsp;b\u003c/strong\u003e highlights the presence of wax-like crystals, as indicated by yellow arrows. A.w – anticlinal wall, P.w – periclinal wall, St – stomata, Tr – trichrome, and W – wax-like structure\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/09629071ffb42744cd9b908c.jpeg"},{"id":104972108,"identity":"23e3458a-dfc2-4f5a-a854-9e5445b4f6e2","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":264705,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence of silicon inclusions visualized with confocal laser scanning microscopy for the adaxial (\u003cstrong\u003ea, e\u003c/strong\u003e) and abaxial (\u003cstrong\u003eb, f\u003c/strong\u003e) surface of leaves in two \u003cem\u003eQuercus robur\u003c/em\u003e trees was grown in shade zone\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ea-d\u003c/strong\u003e – No. 1\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003ee-h\u003c/strong\u003e – No. 2). Silicon has green fluorescence. Fig. \u003cstrong\u003ec′, d′, g',\u003c/strong\u003e and\u003cstrong\u003e h'\u003c/strong\u003e: histograms of the intensity profile of silicon (green line). Ordinate of the histograms indicates the intensity profile of green line, while the abscissa indicates the distance (μm) scanned in \u003cstrong\u003ec, d, g \u003c/strong\u003eand \u003cstrong\u003eh\u003c/strong\u003e; with the this white line illustrating this distance on \u003cstrong\u003ec, d, g \u003c/strong\u003eand \u003cstrong\u003eh. \u003c/strong\u003eBars = 50 μm\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/916296348e225bc1d1510dc1.jpeg"},{"id":104972112,"identity":"8f106046-830b-475d-b05f-20730ce4abf8","added_by":"auto","created_at":"2026-03-19 11:15:09","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":330239,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence of silicon inclusions visualized with confocal laser scanning microscopy for the adaxial (\u003cstrong\u003ea, e\u003c/strong\u003e) and abaxial (\u003cstrong\u003eb, f\u003c/strong\u003e) surface of leaves in two \u003cem\u003eQuercus robur\u003c/em\u003e trees\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ea-d\u003c/strong\u003e – No. 3\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003ee-h\u003c/strong\u003e – No. 4) was grown in sunnier area (without shaded). Silicon has green fluorescence. Figures \u003cstrong\u003ec′, d′, g',\u003c/strong\u003e and\u003cstrong\u003e h'\u003c/strong\u003e: histograms of the intensity profile of silicon (green line). Ordinate of the histograms indicates the intensity profile of green line, while the abscissa indicates the distance (μm) scanned in \u003cstrong\u003ec, d, g \u003c/strong\u003eand \u003cstrong\u003eh\u003c/strong\u003e; with the this white line illustrating this distance on \u003cstrong\u003ec, d, g \u003c/strong\u003eand \u003cstrong\u003eh. \u003c/strong\u003eBars = 50 μm\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/58834a4979204487d2db575f.jpeg"},{"id":105036782,"identity":"576b00b1-d68c-4eaf-833e-46062826e03b","added_by":"auto","created_at":"2026-03-20 07:35:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3215989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/578c788f-d5b3-4c61-a764-7b43a6e58fcd.pdf"},{"id":104972103,"identity":"bb2c2504-66f5-42db-9d8b-c73e60754bf2","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22863,"visible":true,"origin":"","legend":"","description":"","filename":"4TablesNedukha1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/3c9c63eeb5f650f99dbe1523.docx"},{"id":104972106,"identity":"884ea21c-fc88-41e3-a634-7af14195c647","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33280,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemantarymaterials.doc","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/bd09f3fa763a8e12c61133a6.doc"},{"id":104972109,"identity":"4069dcde-d0f1-4116-88fe-b968baf89749","added_by":"auto","created_at":"2026-03-19 11:15:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16564,"visible":true,"origin":"","legend":"","description":"","filename":"Listofpossiblereviewers.docx","url":"https://assets-eu.researchsquare.com/files/rs-8995237/v1/eef1bf7214cbb2bdda692c91.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of microstructure and silicon of leaf in adaptation of Quercus robur trees to different light intensity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStudying the structural and functional organization of \u003cem\u003eQuercus robur\u003c/em\u003e Leaves are crucial for botany and forestry in Ukraine and Europe, as they are a major species forming forests. The practical utilization of oak leaves, bark, seeds, and acorns in phytochemistry and pharmacology adds significant value to this research from both fundamental and applied perspectives. Phenolic acids, terpenoids, and tannins derived from oak exhibit anti-inflammatory, anti-diabetic, and antitumor properties. These compounds hold significant promise as potential drug candidates in the fight against infectious diseases (Taib et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The conservation of oak forests in Ukraine is a growing concern, as climate change brings about more droughts, alongside the impact of atmospheric and soil pollution caused by the war with Russia, which has been ongoing since 2022. Such factors adversely affect the sustainability of oak forests. (Osborn and Taylor \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Novak \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Conserving the common oak is a crucial measure for preserving Europe's and Ukraine's natural landscapes. Additionally, oak stands minimize soil erosion by decreasing sediment Sancho-Napik and enriching the surrounding soil with nutrients (Broome et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tree leaves are often the first to detect environmental changes and respond appropriately. It is known that the morphological traits of oak leaves are adapted to the microclimate of their growth, as demonstrated in previous work on leaf anatomy (Sancho-Knapik et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alonso-Forn et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA wide sample of oak species from around the world has been shown to develop a set of leaf morphological traits with different ecological roles (Mart\u0026iacute;n-S\u0026aacute;nchez et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The outer cell walls of the leaf epidermis serve primarily as both a barrier for penetration and a primary transport route for sunlight, CO₂, and water, while also acting as the interface between plant organs and the environment (Riederer and Schreiber \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Bacelar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; M\u0026uuml;ller and Riedere \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lewandowska et al. 2020). Epidermal walls are where silicon inclusions are synthesized (Wang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and are crucial in regulating sunlight absorption by the surface of the leaf (Mirshafieyan and Gue 2014). The functioning and structure of leaf epidermis rely on temperature, humidity, and UV intensity. The steppes of Ukraine experience temperatures ranging between +\u0026thinsp;22 and +\u0026thinsp;30\u0026deg;C and an average annual rainfall of 400\u0026ndash;600 mm. Trees growing in this area are adapted to severe drought. Don\u0026rsquo;t overlook the fact that they are extensively researched; there are several studies, but natural and human-caused disasters in oak forests are researched extensively. However, there is a lack of understanding about important issues such as the phenotype plasticity of the species and the regeneration of oak stands in the steppe zone of Ukraine.\u003c/p\u003e \u003cp\u003eThis knowledge serves as the foundation for the conservation of oak forests' biological diversity. We assume that the growth of oak trees in southern Ukraine, where conditions feature strong solar radiation and limited water availability, may be attributable to specific traits of leaf epidermal structure withstanding the region's hot climate. In addition, it is known that under conditions of heat, plants synthesize wax in epidermal cells (Reynhardt and Riederer \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Kerstiens \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Rahman et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), as well as amorphous and crystalline silicon (Hiroyuki Takeda et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Grašič et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which can reflect and absorb sunlight, and thus these two components of the epidermal walls can inhibit transpiration processes in leaves, optimizing the water balance of the plant. Therefore, the aim of this study was to investigate the role of the structural-functional signs of the oak leaf epidermis in the adaptation of the species to high solar irradiation in the southern forest-steppe of Ukraine. In addition, the second goal was to compare the role of leaf wax and silicon inclusions in the adaptation of \u003cem\u003eQuercus robur\u003c/em\u003e trees to growth in conditions of strong shading and direct sunlight.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and Environmental metrics\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeaves of common oak, \u003cem\u003eQuercus robur\u003c/em\u003e L. Fagaceae) were collected in june from trees grown in the forest-steppe zone of the Dnepropetrovsk region of Ukraine (48\u0026deg;27\u0026prime;58\u0026Prime;N, 35\u0026deg;01\u0026prime;31\u0026Prime;E, 139 m above sea level). The sampling site of \u003cem\u003eQ. robur\u003c/em\u003e has the following habitat characteristics: 1: A forest of the southern ravine-oak variety grows on the edge of a stand near the village of Bashmachka in the Solonyansky district of the Dnepropetrovsk region, on the right bank of the Dnipro River in Ukraine. At site 2, on the top of a ravine, two oaks (Nos. 1 and 2) grow, while site 1 hosts oaks (Nos. 3 and 4). Refer to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for more information. Specifically, Oak No. 1 grows at the forest edge, Oak No. 2 grows in dense forest, and Oak Nos. 3 and 4 grow alone. In late June, Ukraine's Dnepropetrovsk Region had hot weather. The average daily air temperature in June was significantly higher than normal. For 3\u0026ndash;6 days, the maximum air temperature surpassed 30\u0026deg;C and reached 33\u0026ndash;37\u0026deg;C on the hottest days. The average 10-day air temperature was 1.2\u0026ndash;3.8\u0026deg;C above the multiyear average and ranged from +\u0026thinsp;20.6\u0026ndash;23.0\u0026deg; C (source: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.apk-inform.com/uk/crop/1527945\u003c/span\u003e\u003cspan address=\"https://www.apk-inform.com/uk/crop/1527945\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). There was a precipitation deficit for most days during the ten-day period. High air temperatures and a significant lack of precipitation led to intense moisture loss, particularly in the upper soil layer. The region experienced an ongoing crop drought (source: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.apk-inform.com\u003c/span\u003e\u003cspan address=\"https://www.apk-inform.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). On the day of collection, the weather was sunny. The amount of light on the leaf surface differed between oak trees #1, #2, #3, and #4. PPFD (photosynthetic photon flux density) levels on the adaxial surface of oak #1 leaves were 900 \u0026micro;mol\u0026middot;m\u003csup\u003e-2\u003c/sup\u003e\u0026middot;s\u003csup\u003e-1\u003c/sup\u003e; for oak #2, it was 50 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;; for oak #3 and oak #4, the PPFD was 1500 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;. The LI-250 light meter (USA, LI-COR) was used to measure the PPFD. Light intensity was measured on the upper surface of the lowermost tier of leaf plates for cytological analyses. 15\u0026ndash;20 leaves were collected from each tree's branches, and their length and breadth were measured. To carry out these analyses, we used the centre portion of every other (upper) lobe from five leaves of each type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The leaf area was determined following the procedure outlined by Pandey and Singh (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Each leaf was placed on a sheet of graph paper with millimetre markings, and the outline was traced. The sheet area was then measured. A section of graph paper, equivalent to the leaf's outline, was cut using a paper knife and weighed on an electronic scale. Similarly, one square centimetre of the same graph paper was cut and weighed. Leaves from the same sample were measured multiple times. The area of the leaf was calculated using the non-destructive method with the following equation: Leaf area can be determined using the following formula: Area (square cm) = weight of paper within the outline (g) / weight of paper within the outline (g).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eThe middle portion of each second upper lobe of the leaf plate was fixed in a solution consisting of 2% paraformaldehyde and 1% glutaraldehyde (1:1, vol.) in 0.5 M phosphate buffer (pH 7.2) for 13 hours at +4\u003csup\u003eo\u003c/sup\u003eC. After bringing them to the laboratory, wash the samples with the same buffer and dehydrate them in a series of alcohols (70%, 80%, and 100% ethanol; twice every 30 minutes) as per Talbot and White's (2013) protocol. After dehydrating the samples, they were mounted on on stubs, coated with carbon and gold, and analysed using a JSM 6060 LA scanning electron microscope at 30 kV. Cell size was determined by measuring 30\u0026ndash;40 usual epidermal cells, 30\u0026ndash;35 stomata, and 20\u0026ndash;25 trichomes in three samples from each of the five leaves. All measured variables were then analysed for variance using the Origin 6.1 program.\u003c/p\u003e\n\u003ch3\u003eLaser confocal microscopy\u003c/h3\u003e\n\u003cp\u003eSamples for laser confocal microscopy were prepared following Dabney et al.'s (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) procedure. We exposed sections from the middle of the second (upper) leaf blade of the leaf plate of five leaves from each oak tree (10 \u0026times; 20 mm) to +\u0026thinsp;220\u0026deg;C for three hours until the samples turned grey. The study analysed the presence of silicon in leaf samples employing an LSM5 laser scanning confocal microscope (Zeiss, Germany) with excitation and emission wavelengths of 480 nm and 530 nm, respectively. The study considered five leaves from each tree. The silicon fluorescence intensity was then evaluated using the Pascal program on the LSM5.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eStatistical differences among mean values were determined using the Student\u0026rsquo;s t-test with a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significant differences were observed across all measurements. Origin 6.1 software was employed for data analysis. Each experiment was conducted three times. We measure leaf size, trichome and epidermal cell size, stomata density, and the microstructure of the leaf surface, or the cytochemical study of the fluorescence of silicon inclusions. For cytological research, we chose 20\u0026ndash;22 medium-sized leaves from the lower branches of every oak tree. Ten leaves from each variant were chosen to measure linear dimensions, and five leaves were selected for studying the microstructure of the leaf epidermis and five leaves for conducting cytochemical research on silicon. The average size of the leaf blades for the four oak trees is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The length of the blade, from the apex to the petiole, and the width at the level of the second upper lobe are the first and second values, respectively. The mean value comparison between leaves was done through ANOVA. Origin 6.1 programs performed the analysis of variance for all measured variables.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMicromorphology of\u003c/strong\u003e \u003cstrong\u003eQuercus robur\u003c/strong\u003e \u003cstrong\u003eL. leaves (\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface structure of the leaves of four oak trees was studied. Trees No. 1 and No. 2 grew in the shade in a group of trees on the edge of the forest near the village of Bashmachka in the Dnipropetrovsk Region. Trees No. 3 and No. 4 grew individually on ravine tops in direct sunlight without shade (see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e for the basic parameters of the samples). Leaves from four selected common oak (\u003cem\u003eQ. robur\u003c/em\u003e) trees, regardless of age or growth location, display comparable leaf blade characteristics: the leaves have a short petiole, are hypostomatic (stomata present only on the lower surface of the blade), are elongated-obovate in shape (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), are narrowed downwards, and are pinnately lobed. We found that the leaf blades are rounded and blunt, with shallow notches between them. Some variations in leaf size, area, and ultrastructure were observed in the leaf epidermis. Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e displays the average leaf blade size of the four trees studied.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning electron microscopy (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eLeaves of shaded trees.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eTree No. 1. Adaxial surface\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe oak leaves under study exhibited an irregular upper epidermal surface. The periclinal walls of epidermal cells protruded above the surface. Such walls form spherical or ovoid bulges, around which anticlinal walls produce depressions that cover needle-like and lamellar waxy structures, varying in size from 1 to 4 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, b; Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Stomata and trichomes were absent from the upper epidermis. The periclinal wall protrusions exhibit a partially smooth surface, sometimes coated with wax-like structures, reminiscent of the appearance of the anticlinal recessed walls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e Scanning electron microscopy of adaxial (\u003cstrong\u003ea, b\u003c/strong\u003e) and abaxial (\u003cstrong\u003ec, d, e, f\u003c/strong\u003e) leaf surfaces of \u003cem\u003eQuercus robur\u003c/em\u003e tree No. 1, which was grwn in the shaded zone of Dnipropetrovsk Region, Ukraine. The yellow arrows indicate the location of wax-like crystals. Abbreviations used include A.w - anticlinal wall, P.w \u0026ndash; periclinal wall, St \u0026ndash; stomata, Tr \u0026ndash; trichrome, and W \u0026ndash; wax-like structure\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe abaxial epidermis\u003c/em\u003e. The lower epidermis of oak leaves contains stomata and trichomes, which originate from the lower epidermal cells and the cells of the protruding veins (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). We established that the tested hypothesis regarding the differences in ultrastructural characteristics of leaves including stomata and trichome density in shaded trees and trees under direct sunlight was true. The lower epidermal cells are covered by a layer of lamellar crystalline structures, making it difficult to see and measure their boundaries and sizes. Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e provides details on the density of stomata and trichomes, as well as the average size of stomatal cells and trichomes. The stomata are oval-elongated, with guard cells covered by thin crystalline structures approximately 2 \u0026micro;m in size. While the stomatal gap is free of crystalline structures. Trichomes on the leaf surface are simple, slightly raised, and unbranched, with a drop-shaped base; rounded-plate shaped main parts, and pointed speices. The base of the trichomes is covered by small, stick-shaped crystalline structures, sometimes accompanied by larger, crystal-like structure Trichomes on the leaf surface are simple, slightly raised, and unbranched, with a drop-shaped base, rounded-plate-shaped main parts, and pointed species. The base of the trichomes is covered by small, stick-shaped crystalline structures, sometimes accompanied by larger, crystal-like structures (see Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTree No. 2. Adaxial surface.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe upper surface of leaves from tree number 2 is distinct, with clearly defined epidermal cell contours and an absence of stomata and trichomes (see Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Anticlinal walls protrude above the epidermal surface, forming a tall fence around each cell. The cells on the upper leaf surface are small and have a similar shape to those on tree number 1 (refer to Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The periclinal walls are depressed, and both the anticlinal and periclinal cells are uniformly covered with crystalline structures measuring 1 to 3 \u0026micro;m in size.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOn the abaxial surface\u003c/em\u003e, the epidermal cells are concealed by a continuous layer of lamellar crystalline structures, while stomata and trichomes are present (see Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, d, e, f). The size of the main cells cannot be determined, as seen in sample 1. Additionally, fine crystalline structures that measure approximately 2 \u0026micro;m enclose the stomatal guard cells. The mean density of trichomes is 71\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 per square millimeter of surface area. Trichomes extend from the cells of protruding veins and are positioned above the usual cells. They are simple, unbranched, and slightly raised, with a distal base that is drop-shaped and a middle part that is rod-shaped and pointed at the apex. The trichome base is covered with small, crystalline, rod-shaped structures. The average sizes of stomatal guard cells and trichomes are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeaves of unshaded trees in sun zone.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTree\u003c/em\u003e \u003cstrong\u003eNo.\u003c/strong\u003e \u003cem\u003e3.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdaxial Surface\u003c/em\u003e: An analysis of the leaf ultrastructure of Tree #3 revealed that periclinal walls of the epidermal cells protruded above the surface, creating spherical or ovoid bulges where the anticlinal walls formed depressions. These depressions were covered in needle-like and lamellar waxy structures ranging in size from 1 to 4 \u0026micro;m (refer to Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Stomata and trichomes were absent on the upper epidermis. The surface of the periclinal wall protrusions is partially smooth, sometimes covered with waxy structures, similar to the surface of the anticlinal recessed walls. The cell sizes on the adaxial surface are listed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbaxial Surface\u003c/em\u003e: On the lower surface of the specimen leaf, stomata and trichomes are visible (refer to Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, d, e, f). Trichomes emerge from the pavement cells and from the cells of protruding veins. The stomata are slightly elongated and oval-shaped. The density and size of stomata are listed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Stomatal guard cells are covered with thin crystalline structures measuring about 2 \u0026micro;m, while the stomatal gap is free of such structures. The average size of the stomatal gap measures 18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 x 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;m. The stomatal surrounding pavement cell surface is also coated in a continuous layer of lamellar crystalline structures, which renders the boundaries of the ordinary cells of the lower epidermis indiscernible.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe trichomes are situated above the primary epidermal cells, tilted slightly upward, and occasionally above the veins. The apex base of the trichomes assumes a drop shape; the main part is plate-shaped and crystalline. The lower epidermal usual cells surrounding the stomata and the cells beneath the trichomes are covered by a continuous layer of lamellar crystalline structures. As a result, the borders of the usual cells surrounding the stomata are not visible, which is similar to the structure of the epidermal cells in the leaves of trees No. 1 and No. 2. The trichome base is also covered with small, rod-shaped crystalline structures and occasionally has larger crystal-like structures as well (refer to Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). The average sizes of stomatal closure cells and trichomes are provided in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeaves of tree\u003c/em\u003e No. \u003cem\u003e4.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdaxial Surface\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ultrastructure analysis of the adaxial leaf surface of Tree No. 4 revealed the absence of trichomes and stomata. The epidermis surface was composed of typical epidermal cells, with periclinal walls projecting above the surface and anticlinal walls recessed (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e\n\u003cp\u003eThe spherical or ovoid bulges protruding from the periclinal walls of the epidermal cells resulted in depressions formed by the anticlinal walls. These depressions were covered in needle-like and lamellar wax structures of varying sizes, ranging from one to four \u0026micro;m. Both the anticlinal walls and parts of the periclinal walls were covered in waxy structures. Stomata and trichomes were not present on the upper epidermis. The periclinal wall projections of most cells have a smooth surface, although some periclinal walls and the surface of the anticlinal recessed walls are occasionally covered with waxy structures. Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e presents the cell size on the adaxial surface.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbaxial Surface\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leaves of Oak No. 4 exhibit stomata and trichomes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec, d, e, f) that originate from the main epidermal cells and the vein cells. The stomatal guard cells are coated with needle-like waxy structures that measure approximately 2 \u0026micro;m. The stomatal gap lacks crystalline structures with an average size of 17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 x 5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026micro;m. The lower epidermal cell surface that encircles the stomata features a constant waxy layer in diverse forms\u0026mdash;needle-like,lamellar, and crystalline. Trichomes sit atop the main epidermal cells, being slightly lifted and occasionally positioned over the veins. They are simple, raised, and unbranched with an elongated or oval base and a rod-shaped head. The trichome base is encompassed with needle-like waxy structures. The boundaries of the usual cells in the lower epidermis are rendered invisible by the continuous layer of lamellar crystalline structures that cover the surface of the epidermal cells surrounding the stomata, a feature consistent with previous specimens. The average size of stomata cells and trichomes can be found in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaser Confocal Microscopy of Silicon in\u003c/strong\u003e \u003cstrong\u003eQuercus robur\u003c/strong\u003e \u003cstrong\u003eleaf epidermis (\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeaves of tree No. 1 grown in shade zone.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur study shows the bright green fluorescence of silicon inclusions in the epidermal cells of oak tree No. 1 leaves (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). The fluorescence of the silicon was detected in the anticlinal and periclinal walls of the adaxial epidermis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eThe silicon inclusions were densely arranged in the anticlinal walls, forming nearly continuous layers. Individual silicon inclusions are visible in the periclinal walls. Silicon fluorescence is noticeable in the walls of usual epidermal cells, guard cells of stomata, and at the base of trichomes in the lower epidermis. The silicon had a single, individual inclusion in its form (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). The intensity of the silicon fluorescence profile varied among the studied cells and depended on the leaf surface and cell type (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec, c\u0026apos;, d, d\u0026apos;). The silicon intensity varied between the anticlinal and periclinal walls of epidermal cells on the adaxial surface. The intensity in anticlinal walls was 120\u0026thinsp;\u0026plusmn;\u0026thinsp;13 relative units, while in periclinal walls, it was significantly lower, quantifying to 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 relative units. The intensity of silicon in the anticlinal walls of ordinary cells of the lower epidermis was 75\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 relative units. In contrast, in the periclinal walls of those cells, it was 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3, and in trichomes and the closing cells of stomata, it was 156\u0026thinsp;\u0026plusmn;\u0026thinsp;11 and 71\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 relative units, respectively. The silicon inclusions are most densely concentrated in the anticlinal walls of the adaxial epidermis and in the trichomes of the abaxial epidermis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeaves of tree No. 2 grown in shade zone.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis showed green silicon fluorescence in the cell walls of epidermal cells in both the upper and lower epidermis, as well as in stomata and at the base of trichomes in \u003cem\u003eQ. robur\u003c/em\u003e leaves of tree No. 2, which were grown in shade (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). Additionally, silicon fluorescence was detected in the anticlinal and periclinal walls of the main epidermal cells of the adaxial epidermis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg). The relative units of the fluorescence intensity were 105\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0 and 55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3, respectively. Silicon fluorescence was also found in the guard cells of stomata, at the base of trichomes, and in the anticlinal and periclinal walls of epidermal cells in the abaxial epidermis, consistent with the previous sample. The intensity of the silicon profile varied depending on the leaf surface and cell type, as shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg, g\u0026apos;, h, h\u0026apos;. Silicon levels were 80\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 in anticlinal walls, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 in periclinal walls, 121\u0026thinsp;\u0026plusmn;\u0026thinsp;12 in trichomes, and 65\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 in stomata, all reported in relative units. In summary, the periclinal walls of the primary cells on the upper and lower surfaces, including the walls of stomata, have low profile intensity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeaves of tree No. 3 grown in sunnier area.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA cytochemical analysis of silicon in \u003cem\u003eQ. robur\u003c/em\u003e leaves was conducted on tree No. 3, which grew in direct sunlight without shade. The results revealed green fluorescence of silicon in the walls of upper epidermal cells, trichomes, stomata, and conducting bundle cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, g, h).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eSilicon fluorescence was also observed in various parts of the leaf, including the abaxial epidermis cells, guard cells of stomata, and trichome base. The intensity of the silicon profile varied depending on the leaf surface and cell type, measured in relative units. The highest intensity was found in the anticlinal and periclinal walls of the upper epidermis, with values of 140\u0026thinsp;\u0026plusmn;\u0026thinsp;12 and 124\u0026thinsp;\u0026plusmn;\u0026thinsp;11 relative units, respectively. Similarly, the intensity of the profile was high in the anticlinal walls of the lower epidermis, at the base of trichomes and stomata guard cells, with values of 100\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7, 180\u0026thinsp;\u0026plusmn;\u0026thinsp;15, and 125\u0026thinsp;\u0026plusmn;\u0026thinsp;12 relative units, respectively. However, the periclinal walls of lower epidermal cells showed a lower profile intensity of 80\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 relative units (See Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec, c\u0026apos;, d, d\u0026apos;).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeaves of tree No. 4 grown in sunnier area..\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cytochemical analysis revealed green fluorescence for silicon within the walls of ordinary epidermal cells, trichomes, stomata, and conducting bundle cells. In the adaxial epidermis, silicon fluorescence was detected in the anticlinal and periclinal walls of the upper epidermal cells, similar to the previous sample (see Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). Silicon fluorescence was also detected in the anticlinal and periclinal walls of the abaxial epidermal cells, guard cells of stomata, and trichome bases. The intensity of the silicon profile varied based on the leaf surface and cell type, as indicated in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg, g\u0026apos;, h, h\u0026apos;. The highest silicon profile intensity was in the anticlinal and periclinal walls of the upper epidermis, with relative units of 145\u0026thinsp;\u0026plusmn;\u0026thinsp;13 and 115\u0026thinsp;\u0026plusmn;\u0026thinsp;12, respectively. Additionally, significant profile intensity was found in the anticlinal walls of the lower epidermis, trichomes, and stomatal guard cells, with values of 120\u0026thinsp;\u0026plusmn;\u0026thinsp;11, 185\u0026thinsp;\u0026plusmn;\u0026thinsp;12, and 128\u0026thinsp;\u0026plusmn;\u0026thinsp;11 relative units, respectively. Conversely, the periclinal walls of ordinary cells in the lower epidermis demonstrated lower profile intensities, measuring just 95\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7 relative units.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have studied the leaves of \u003cem\u003eQ. robur\u003c/em\u003e trees growing in different conditions in the forest-steppe zone (both in the forest and on the slopes of ravines) of the Dnepropetrovs'k region of Ukraine. We found similarities and differences in leaf blade size, leaf surface ultrastructure, localization, and content of silicon inclusions in the studied tree leaves depending on the light intensity of the environment where the trees grewn in sunnier or shade zones. Furthermore, we found that the tested hypothesis about the differences in leaf size between shaded trees and trees in direct sunlight was correct. The leaf blades of the first two trees, located on the forest edge and shaded by other trees (trees No. 1 and No. 2), were significantly larger than those of trees No. 3 and No. 4 growing without shade on ravines. The leaf, being the most adaptable organ to environmental changes (Nevo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), shows the effects of environmental factors more clearly than stems and roots. Smaller leaf sizes can result in reduced water loss, especially from the adaxial surface. Such a phenomenon has been observed in the leaves of two-year-old saplings of \u003cem\u003eQuercus petraea\u003c/em\u003e and \u003cem\u003eQ. robur\u003c/em\u003e, as reported by Dupouey and Dreyer (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and in \u003cem\u003eOlea europaea\u003c/em\u003e trees growing in environments with high air temperatures and low water supplies, as found by Bacelar et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Plants typically respond to shade by producing larger leaves with a lower mass per unit area, allowing for more efficient light capture per unit mass, as noted by Niinemets and Sack (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results of studying the size of oak leaves showed that oak trees growing in direct sunlight are characterized by small leaves. Our data are confirmed by other researchers. It is well-established that trees growing in sunnier areas produce smaller leaves with greater mechanical strength (Onoda et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, these leaves have thicker cuticles and epicuticular wax layers. This phenomenon is also observed in the leaves of succulent and mesophytic plants (Richardson and Berlyn \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lewandowska et al. 2020). The unshaded leaves of plants that grow in direct sunlight are characterized by an increase in vessel and stomata density compared to shaded leaves (Karabourniotis and Bornman, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Karabourniotis et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Granier and Tardieu (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) determined that the size of unshaded leaves is significantly affected by light intensity, which can impede cell division and elongation. These authors have shown that the rate of cell division and rate of cell elongation decreased with increased illumination. This phenomenon can be attributed to shading as a prevailing abiotic stressor, given its occurrence as a result of light obstruction by neighboring plants.\u003c/p\u003e \u003cp\u003eOur research indicated that shaded oak trees (No. 1 and No. 2) have fewer simple non-glandular trichomes in their leaves compared to oak trees growing in non-shaded conditions (No. 3 and No. 4). The increase in trichome density in the unshaded oak trees (No. 3 and No. 4) is likely due to their role in protecting the leaves from environmental stressors. Simple elongated trichomes have been found in the leaves of various tree and grass species, including \u003cem\u003eQ. laevis\u003c/em\u003e, red oak (\u003cem\u003eQ. rubra\u003c/em\u003e) (Kryvoruchko and Bessonova \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and black walnut (\u003cem\u003eJuglans nigra\u003c/em\u003e) (Prasad and G\u0026uuml;lz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Wagner et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; LoPresti \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Oksanen \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The increase in trichome density in unshaded oaks is thought to be related to their function as a barrier against UV radiation, increased transpiration, and pathogens (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These structures are crucial for the development of organs and are supported by phenolic compounds, specifically flavonoids. The formation of trichomes and the accumulation of phenolic compounds are linked at the molecular level. As non-glandular trichomes develop, they show significant morphological similarities to glandular trichomes. As they continue to grow and the secondary wall thickens, phenolic substances are moved to the cell walls of the trichomes (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; LoPresti \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTrichomes act as optical filters, protecting sensitive tissues from UV radiation by screening out harmful wavelengths through the scattered deposition of phenolics in the cell walls. The increased light reflectance on the surface provides protection against strong visible radiation (Skaltsa et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Karabourniotis et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our research, in conjunction with the aforementioned publications, led us to conclude that the leaf trichomes of oaks No. 3 and No. 4 protect the epidermis from high solar irradiation and reduce the intensity of cuticular transpiration. We observed that the intensity of illumination did not affect the presence of wax-like inclusions on the cell walls of the epidermis, the closing cells of stomata, and the base of trichome on the adaxial and abaxial surfaces of leaves from four \u003cem\u003eQ. robur\u003c/em\u003e trees in Ukraine's forest-steppe zone. Wax can exist in cells in two forms: bound to the cuticle or free, creating crystals of different shapes on the cell surface (Kerstiens \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The wax serves as a crucial barrier against unregulated water loss in leaves, has the capability to absorb or reflect UV radiation, and suppresses cuticular transpiration (Riederer and Schreiber \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The presence of waxes has been noted in the leaves of different tree species (Prasad and G\u0026uuml;lz \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Prasad et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The simultaneous presence of crystalline, liquid, and amorphous wax phases could be distinctive characteristics of various organs of numerous plant species (Reynhardt and Riederer \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; M\u0026uuml;ller and Riederer 2005).\u003c/p\u003e \u003cp\u003eThe permeability of the amorphous phase is determined by the degree of crystallinity of the wax, which affects the effectiveness of the diffusion barrier. The study concludes that epicuticular waxes remain on the leaf surface and limit access to the pores in the cuticle. Based on the data regarding the role of wax on leaf surfaces and our experiment results on the existence of wax-like structures on the anticlinal walls of the upper epidermis and on all types of cells of the abaxial epidermis of leaves from four \u003cem\u003eQ. robur\u003c/em\u003e trees, we can deduce the following suggestion: Crystalline wax-like structure is a structural hallmark of oak leaves, regardless of the light intensity, and it regulates transpiration and optimizes the leaf blade's water balance.\u003c/p\u003e \u003cp\u003eWe revealed the presence of silicon in leaves of investigated oak trees. It is known that silicon in plant cells functions as a biological lens, helping incident infrared light penetrate efficiently inside cells and leaves when combined with carbohydrates, proteins, or lipids in the cell walls (Guerriero et al., 2016; Grašič et al., 2020). These properties can facilitate efficient heating of plants as absorbed far-infrared light is converted into heat (Ma et al., 2011; Hiroyuki Takeda et al., 2013). Silicon inclusions (8\u0026ndash;12 \u0026micro;m in size) in the epidermis effectively cool leaves via highly efficient thermal radiation in the mid-infrared range, according to Wang et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Stomatal transpiration also cools leaves. Reduction of transpiration in plants can be achieved by depositing silica structures in the leaf epidermis. This approach may be practical and environmentally friendly for improving plant resistance to high temperatures. The results indicate that plants infused with biosilicon and subjected to temperature stress employ an uncomplicated physical strategy to defend against heat stress.\u003c/p\u003e \u003cp\u003eSilicon is capable of absorbing light fluxes ranging from infrared to ultraviolet. In plants, the refractive index of silicon structures is approximately equal to the wavelength of visible light. As a result, not only do such structures absorb light, but they also reflect it in the region of wavelengths referred to as the photonic band gap (Skaltsa et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Large \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Yahaya et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This optical phenomenon is due to the slowing down of light's group speed at the edges of the photon zone. In this scenario, the interaction between light and materials is intensified. Periodic silicon structures facilitate the enhancement of light reflection and absorption, while higher plants possess characteristic structures that sense infrared or far-infrared radiation. Pedrotti and Leno (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) indicate that the radius of curvature and size of an object are significant parameters for light reflection. Sunbeams can remain unaltered in direction based on the thickness and curvature of the lens. The periclinal walls of the upper epidermis of oak leaves, which we examined, exhibited diverse shapes, either convex or concave depending on the degree of illumination. We were able to identify them as a convex lens (oaks No. 1, 3, and 4), a concave lens (oak No. 2), or a cylindrical lens, which corresponds to the anticlinal walls of the epidermis of all examined samples. The question of UV ray transmission, absorption, and reflection intensity through cell walls acting as lenses remains unanswered and requires further study by biophysicists.\u003c/p\u003e \u003cp\u003eResearchers recently discovered silicon-rich trichomes on both the upper and lower leaf surfaces of the \u003cem\u003eAphananthe aspera\u003c/em\u003e (Muku tree) in Osaka, Japan (Hiroyuki Takeda et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The team used infrared spectroscopy to analyze the trichomes and found that they play a significant role in absorbing far-infrared light due to specific chemical bonds in organic materials and silica. Our research in southern Ukraine confirmed the presence of silicon inclusions in the upper and lower leaf epidermal cells of all oak trees surveyed. The magnitude of the silicon profile varied depending on the type of epidermis and cell. Observations showed that oak trees grown in direct sunlight had greater silicon fluorescence in the cell walls of trichomes, stomata, and upper and lower epidermal cells. This led to the conclusion that increasing silicon content in these areas enhances sunlight absorption and reflection, optimizing photosynthesis and thermal stability.\u003c/p\u003e \u003cp\u003eOur data show that the use of leaf blades of oaks growing under direct solar irradiation is promising for the use of biological forms of silicon, which has potential applications in medicine, pharmacology, and agriculture. Plants with high silicon content, such as oak trees, have been found to be beneficial for bone tissue restoration in medicine (Taib et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, the use of oak leaves as fertilizer has shown positive results in agriculture, particularly in improving plant health during water stress conditions. The application of oak leaves as fertilizer in agriculture yields positive results by optimizing the researchers found that adding oak leaf powder and biofertilizer to soil improved the growth and biochemical composition of tomato leaves of several genotypes under water stress conditions (Nawroz Abdul-Rezzak et al. 2022). In conclusion, this study highlights the potential benefits of oak leaves for mitigating water stress and improving plant life, making them promising for practical applications.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe study of the leaves of \u003cem\u003eQuercus robur\u003c/em\u003e trees grown in sunnier areas or shade zones in the southern forest-steppe zones of Ukraine revealed phenotypical plasticity and adaptive structural signs of oak leaf blades to changes in sunlight lighting. These differences, such as leaf size, density of trichomes and stomata, and a bulge in the periclinal cell walls in leaf epidermis, may reflect adaptations to the intensity of sunlight. These features help reflect sunlight from the upper surface of sunlit oak leaves compared to shaded leaves. The presence of such structural features shows that oak leaves are characterized by the ability to adapt to high solar irradiation and reserve optimal water balances in the leaves. The presence of such structural features shows that oak leaves are characterized by the ability to adapt to high solar irradiation and reserve optimal water balances in the leaves.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eA laser confocal microscopic study of silicon inclusions present in the leaf epidermis of \u003cem\u003eQ. robur\u003c/em\u003e revealed that the external environment\u0026mdash;sunnier areas or shade zones\u0026mdash;can change the content of silicon in the leaf epidermal wall. Leaves of trees grown in strong solar irradiation contained significantly higher values of silicon inclusions in cell epidermis. We assume that an increase in the silicon content in the epidermis of oak leaves growing without shade leads to a reduction in cuticular and stomatal transpiration and optimization of the water balance of oaks growing in direct sunlight, which helps trees adapt to the climate and aridization under the action of strong solar irradiation. epidermis,\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe author is grateful to Dr. \u0026nbsp;Shevchenko G. \u0026nbsp; for help during collecting samples for the study, and \u0026nbsp;Dr. Topchiy N. for help during measure the sun intensity on the tree leaves' surface.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e. The study was financially supported by the National Academy of Science of Ukraine; the studies were performed in the Department of Cell Biology and Anatomy of Institute of Botany of National Academy Sciences of Ukraine; №\u0026nbsp;8-22, 2022-2023 ,state registration number; title of \u0026nbsp;project -\u0026nbsp;Determination of structural-functional and molecular features of resistance of common oak (\u003cem\u003eQuercus robur\u003c/em\u003e L.) to aridization of the Ukrainian climate\u0026nbsp;(2022-2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp; \u0026nbsp;The manuscript is not published elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe author declares that she has no conflict of \u0026nbsp;interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp; Dr Nedukha Olena designed the re­search methodology, \u0026nbsp;fixed and photographed samples, examined those using electron and laser confocal microscopy, described the results, and wrote a manuscript of the article collected and analyzed the data, and drafted the manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlonso-Forn D, Sancho-Knapik D, Fari\u0026ntilde;as MD, Nadal M, Mart\u0026iacute;n-S\u0026aacute;nchez R, Perrio JP, Resco de Dios V, Peguero-Pina JJ, Onoda Y, Cavender-Bares J, \u0026Aacute;lvarez-Arenas TEG, Gil-Pelegr\u0026iacute;n E (2023) Disentangling leaf structural and material properties in relationship to their anatomical and chemical compositional traits in aks (\u003cem\u003eQuercus\u003c/em\u003e L.). 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Optics Express 21:5924\u0026ndash;5930. https://doi.org/10.1364/OE.21.005924\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leaf micromorphology, Quercus robur, Epidermis structure, Light intensity","lastPublishedDoi":"10.21203/rs.3.rs-8995237/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8995237/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe microstructure, localization, and content of silicon inclusions in the leaf epidermis of \u003cem\u003eQuercus robur\u003c/em\u003e trees grown in forest-steppe zones of southern Ukraine with different sunlight intensities were studied with the electron microscopic method and laser confocal microscopy. We established the influence of sun intensity on silicon content in oak leaf epidermis. It was found that silicon is most accumulated in trichomes, stomata, and ordinary epidermal cells of oak leaves. The observed phenotypic plasticity of leaves is manifested in an increase in the trichome number and stomatal density, as well as in a decrease in leaf size and area in trees growing in direct sunlight. Common oaks from the southern region of Ukraine are able to grow normally and adapt to high levels of solar radiation due to their increased ability to absorb and reflect light by leaf surface. The various characteristics of these leaves can be used for practical applications.\u003c/p\u003e","manuscriptTitle":"Role of microstructure and silicon of leaf in adaptation of Quercus robur trees to different light intensity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 11:14:58","doi":"10.21203/rs.3.rs-8995237/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d54d1b8d-73e2-48c8-b028-ca839ddd138e","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T15:10:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 11:14:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8995237","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8995237","identity":"rs-8995237","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}