Influence of urban environment factors on morphometric parameters and accumulation of secondary metabolites in Cercis canadensis L. and Cercis siliquastrum ‘Alba’

Green spaces in the urban greening system play a crucial role in the landscape and architectural, sanitary and hygienic, utilitarian and decorative, and other aspects of urban autecological relations. In particular, all green spaces reduce the level of gas and dust pollution, improve the microclimate by reducing the ambient temperature and increasing humidity, but different species cope with these tasks in different ways, so the question arises of the correct selection of plants for urban landscaping in accordance with their environmental characteristics.

Due to climate change and a significant increase in anthropogenic impact, it is necessary to expand the range of planting materials when creating urban plantings. As noted by N. Sey- idoglu Akdeniz (2020), the use of plants of the genus Cercis L. for urban landscaping with white, pink, and purple colours gives a sense of calm, continuity and vitality, which, in turn, plays an important role in the recreation of urban populations.

Results of the study by N. Nuzhyna et al. (2022) on determining plant drought resistance, based on the indicators of leaf water content and moisture loss per unit time, indicate that Cercis canadensis L. is quite drought-resistant. The paper reveals new aspects of plant adaptation to changing environmental conditions, and considers strategies for plant survival in a rapidly changing climate. According to A. Ne- jad et al. (2018), lead exposure at a dose of 30 mg/l had a negative effect on the development of C. siliquastrum L.: the area of the leaf blade, the length of shoots, and the water content in the leaves decreased. However, this effect was offset by the use of nitrates (100 mg/l). These results indicate the importance of studying plant interactions with toxic metals and possible measures to reduce their exposure. According to F. Vigno- li et al. (2021), the use of lawns alone reduces the temperature of the asphalt surface by 0.5°C, and in combination with trees and shrubs - by about 0.9°C, which reduces the negative impact of "heat islands" in the urban environment. In addition, such an ecosystem approach to the arrangement of lawns, trees and shrubs contributes to improving air quality and the overall urban climate. Green spaces help absorb water and reduce the risk of flooding, helping to create a more sustainable and environmentally balanced environment. These positive properties of green spaces confirm the importance of their implementation for achieving sustainable development and improving the quality of life in cities.

Moreover, according to H. Bahri (2021), concentration of flavonoids in flowers of Cer- cis siliquastrum L. decreases as they develop, and the content of terpenes, on the contrary, increases, which may indicate changes in secondary metabolism depending on the phase of plant development. These detected changes in the chemical composition of flowers of Cercis siliquastrum L., in particular, reduction in the concentration of flavonoids and increase in the content of terpenes may also be of practical importance in the context of using these plants in landscape design or urban landscaping, based on their characteristics and adaptive properties. Given that changes in secondary metabolism may be associated with growth and develop - ment phases, further study of these mechanisms may help develop strategies to optimise growing conditions of Cercis siliquastrum L. to increase the biological activity and stability of these plants in various environmental conditions.

The study by A. Delbari et al. (2019) on the absorption of cadmium and lead by the roots of two plant species of the genus Cercis L. helped to identify the difference between them in resistance to soil contamination with toxic metals. It is evident that Cercis siliquastrum L. exhibit various reactions to the presence of these toxic metals, which may be of practical importance for their use in phytoextraction or phytostabilisation programmes of contaminated soils. Consideration of these interactions can be important for sustainable management of the urban environment and the effective use of plants in phytoremediation processes.

The purpose of the study is to investigate the bioecological features of plants of the genus Cercis L. and the correlation between morphometric and biochemical parameters.Materials and Methods

The research was conducted between January 2023 and September 2023.

Conditions for the growth of research objects. The selected research objects were located in Kyiv, in particular, in the Shevchenkivskyi district (Fig. 1). The city's climate is moderately continental, with mild winters and rather hot summers. The average temperature in July and January was +22°C and -2.8°C, respectively (Osadchyy et al., 2013).

Figure 1. Layout of Cercis L. plantations in Kyiv

Source: compiled by the authors

The habitats of all plants can be conditionally divided according to the influence of external factors, such as air pollution, solar insolation, and soil salinity. For example, plants growing in the O.V. Fomin Botanical Garden were exposed to an average level of insolation (the amount of direct sunlight is about 6 hours per day) and air pollution, and the absence of soil salinity. Trees growing near the Beresteyska metro station had a high level of insolation (direct sunlight for about 10 hours a day) and were exposed to high levels of air pollution because they were located on the southern border of the Nyvky park. The plants growing on Tabirna Street were in shady conditions (the amount of direct sunlight was about 3 hours per day). The level of salinity was determined conditionally, depending on the distance of plants from pedestrian paths or highways. In particular, the plants growing in the park on Kirponos Street were characterised by a high level of insolation and soil salinity, as the plants grew in close proximity to the roadway and pedestrian path.

During the survey of plants of the genus Cercis L., their condition was evaluated on a 5-point scale for assessing the condition of woody plants in outdoor plantings according to the method (Kuznetsov et al., 2000):

¦ 5 points (trees without suppressed growth, with a full leaf surface);

¦ 4 points (trees with growth that generally correspond to the norm and with approximately 20-25% of the inactive surface);

¦ 3 points (trees with weakened growth, which have about 50% of the inactive leaf surface);

¦ 2 points (trees with suppressed growth, the growth rate of current growth is almost nonexistent, have about 75-80% of the inactive leaf surface);

¦ 1 point (dead and drying, without current growth of trees with 100% inactive leaf surface).

Morphometric method. The analysis of morphometric parameters of shoots was carried out to assess the features of plant growth processes depending on the growing conditions. Sampling was carried out in 5 localities. A total of 15 plants were used in the experiment. The length and diameter of internodes of annual shoots of 14 C. canadensis L. plants and shoots of 1 C. siliquastrum `Alba' plant (n = 75) were measured.

Secondary metabolite profiling method. The separation of substances was performed by high-performance thin-layer chromatography (HPTLC) on Silicagel G 60 (Merck) plates in a solvent system: ethyl acetate-formic acid-acetic acid-water (v/v/v/v - 100:11:11:25). The resulting chromatograms were treated with 0.5% NP reagent in ethyl acetate, followed by treatment with 1.0% PEG (polyethylene glycol) 400 and heating at 90°C for 1 min. Flavonoids and other polyphenols on the chromatogram were detected in ultraviolet light (X max = 365 Nm). The Rf (retention index) values of individual compounds were determined photodensito- metrically using the Sorbfil TLC Videodensitometer computer programme.

Autofluorescence of shoot tissues was studied on an inverted microscope using a multichannel fluorescent imaging system (EVOS FL System, ThermoFisher Scientific, USA).

Method of statistical data processing. The measurement results are presented as the average value ± standard error (x±SE). The significance of the difference (p<0.05) between the obtained data was determined by variance analysis (one-way ANOVA) using Tukey's post-hoc test in the XLSTAT software suite (Addinsoft Inc., USA, 2010). Principal component analysis (PCA) and cluster analysis were performed in the XLSTAT software suite. Sigma Plot 12.0 (Systat Software, Inc.) was used for regression analysis.

Object of study - the object of study is the external state of plants C. canadensis L. and C. siliquastrum `Alba' and their sensitivity to urban environmental factors. The study was conducted in compliance with the Convention on Biological Diversity (1992) and the Convention on the Trade in Endangered Species of Wild Fauna and Flora (1973).

Annual shoots were collected from plants growing within the city of Kyiv. Sample preparation included cleaning of dirt and removal of unnecessary plant residues, followed by determining metric parameters (internode length and diameter). The parameters were measured using a caliper and the results were recorded in Microsoft Excel, followed by analysis of the data.

Results

Cercis canadensis L. - a tree up to 18 m high, with a spreading crown and a black and grey crust, sometimes it is a large shrub, which makes it attractive and versatile in terms of use in landscape compositions (Fedorovskyi et al., 2013). Young shoots are reddish-brown. The leaves are cordate and rounded, 6-12 cm wide. Like the European species, it blooms before the leaves open in May. Flowers are pink to purple in colour, 1.0-1.2 cm long, in bunches on branches or on trunks (cauliflory - development of flowers from dormant buds on the trunk), very decorative (Domshyna & Shcherbak, 2015). Its natural habitat is North America. It most often grows on rich soils in lowlands and near marshes, and is moisture-loving. It is found in undergrowth, in valleys on the edges, in sparse forests. In the conditions of Kyiv, it regularly flowers and bears fruit (Kalinichenko, 2003; Zayachuk, 2014)

The general condition of the plantings varies: the condition of the plantings growing near the Beresteyska metro station was approximately 35 years old and rated at 4 points (dry branches, trunk damage or stunted development); trees growing on Tabirna Street were approximately 15-20 years old and rated at 3 points; the condition of plantings growing on Kirponos Street and the territory of the Institute of Horticulture of the NAAS of Ukraine was approximately 15-25 years old and rated at 4-5 points. The condition of C. canadensis L. and C. siliquastrum `Alba' growing on the territory of O.V. Fomin Botanical Garden is rated as "good" - there is a small number of dry branches and the trees are about 40-45 years old and 20 years old, respectively (Fig. 2). Detailed evaluation information for each tree is provided in Table 1.

Figure 2. General view of plantings of Cercis canadensis L.

Note: a - group of plants near the Beresteyska metro station; b - a plant on Tabirna Street; c - group of plants on Kirponos Street; d - cauliflory phenomenon

Source: photo by the authors

Table 1. Condition and location coordinates of the plants under study

Note: *Cercis siliquastrum `Alba'; NAASU - National Academy of Agrarian Sciences of Ukraine Source: compiled by the authors

As can be seen from the regression models below (Fig. 3), the peaks of the curves are shifted to the right, which may indicate that the plants do not have enough sum of active temperatures to complete the processes of lignification of shoots and a full transition to winter. In addition, during the observation of plants during the growing season, it was found that Cercis canadensis L. is characterised by the separation and falling of one or more of the last internodes.

Figure 3. Dynamics of changes in the length of internodes of annual shoots during the growing season

Source: developed based on the authors' research

A decrease in the approximation coefficients of the Gaussian model describing the dynamics of changes in the length of internodes is associated with a shift of the maximum growth peak to the right in the time coordinate. Indicators of the length and diameter of internodes of annual shoots and their ratio, depending on the place of plant growth, are shown in Table 2.

Table 2. Morphometric parameters of plants Cercis L. (x±SE, n=5)

Note: A - Beresteyska metro station; B - Tabirna Street; C - Kirponos Square, D - Institute of Horticulture of NAAS of Ukraine; E - O.V. Fomin Botanical Garden (E1 - Cercis siliquastrum `Alba'; E2 - Cercis canadensis L.); where "1", "3", "5" - first, middle, and last internodes

Source: compiled by the authors

Table 2 shows an inverse relationship between the length of the internode and its diameter. For plants growing in conditions of shading or high humidity, this ratio increases, which is generally typical for plants that usually grow in lighted areas. It was also found that in Cer- cis L. plants, probably due to the insufficient amount of the sum of active temperatures, the process of development of a separate cell layer begins, which blocks the transport system and the supply of nutrients to the top of the shoot (Fig. 4). This process was more active in plants in sunny areas, possibly due to the more intensive growth of annual shoots.

Figure 4. Process of separation of the last internodes on the tops of shoots of Cercis canadensis L.

Note: a - upper internode separation zone

Source: developed by the author

In the places of "scarring", and living parts of the internode and those that began to die, the development of a layer of cells similar to the periderm was observed. The overgrown periderm thickens in the area of the conductive bundles, which leads to a blockage of nutrient supply to the shoot apex. As a consequence of this process, the last internodes die off. Hypothetically, this process is genetically determined and is aimed at protecting the apical meristem during the dormant period of plants in winter (Fig. 5).

Figure 5. Autofluorescence of annual shoot node tissues in the zone of separating layer formation (radial section)

Note: a - cortex; b - phloem; c - xylem; d - core parenchyma; e - separation layer; Ap - apical part; Bs - basal part; DAPI (Xexc=357/44 nm; Xem=447/60 nm); GFP (Xexc=482/25 nm; Xem=524/24 nm)

Source: developed based on the authors' research

Biochemical profiling of secondary metabolites of periderm and bark of annual plant shoots of Cercis canadensis L. identified 15 substances that, according to Rf indicators and the specifics of autofluorescence in ultraviolet (UV) (3=365 Nm), belong to phenol carboxylic acids, their conjugants and flavonoids. Based on the results of cluster analysis, according to the characteristics of phytochemical profiles, 3 main groups of plants were identified. For plants that have developed the first cluster, it is characteristic that they grow in sunny areas without signs of shading, but there are no substances with Rf~0.69 and Rf~0.77. Plants that are combined according to the biochemical profile in the second cluster are characterised by the presence of flavonoids with Rf~0.69 and Rf~0.77. Externally, these plants have no signs of growth suppression, without much damage to the trunk or tree as a whole. 3 out of 5 plants grow in a group planting, other plants also grow in a sunny area, and their age is in the range of 25-35 years. In addition, the plant under the code S5 is characterised by a fairly high concentration of the substance Rf~0.69 and Rf~0.77. This plant developed chlorophyll-deficient leaves during the growing season. There may be a certain relationship between the increased concentration of substances and depigmentation of the leaf plate, which requires additional research (Fig. 6).

Figure 6. Chlorophyll-deficient (a) and normal (b) leaf

Source: developed by the authors

The third cluster was divided into 2 subclusters. In one of them, a substance with an Rf of~ 0.39 was found, which was not present in any sample of the bark and periderm of annual shoots. A reduced amount of phenolic compounds on the chromatogram is typical for plants that are united in the second subclaster of the third cluster.

Figure 7. Results of cluster (AHC) (a) and biplot principal component analysis (PCA) (b)

Source: developed by the authors

These plants grow in shaded conditions and in drafts. Given a direct relationship between the lighting mode and the synthesis of phenols, phenolic compounds play an important role in protecting plants from ultraviolet radiation. And the synthesis centres of some of these substances are partially connected with chloroplasts. This explains the reduced concentration of substances in the periderm and bark of plants of this subclaster.

Based on the results of biplot analysis based on principal components (PCA), it was found that the total variance for the main components F1 and F2 is 44.68% (Fig. 7). According to the first main component, substances with a significant contribution to the total dispersion of Rf~ 0.97; 0.37 and Rf~ 0.93; 0.27 are distinguished.

Discussion