*Article* **Exogenous Application of Chitosan Alleviate Salinity Stress in Lettuce (***Lactuca sativa* **L.)**

**Geng Zhang 1,2,†, Yuanhua Wang 1,2,†, Kai Wu 3, Qing Zhang 1,2, Yingna Feng 1,2, Yu Miao <sup>1</sup> and Zhiming Yan 1,2,\***


**Abstract:** Soil salinity is one of the major factors that affect plant growth and decrease agricultural productivity worldwide. Chitosan (CTS) has been shown to promote plant growth and increase the abiotic stress tolerance of plants. However, it still remains unknown whether the application of exogenous CTS can mitigate the deleterious effects of salt stress on lettuce plants. Therefore, the current study investigated the effect of foliar application of exogenous CTS to lettuce plants grown under 100 mM NaCl saline conditions. The results showed that exogenous CTS increased the lettuce total leaf area, shoot fresh weight, and shoot and root dry weight, increased leaf chlorophyll a, proline, and soluble sugar contents, enhanced peroxidase and catalase activities, and alleviated membrane lipid peroxidation, in comparison with untreated plants, in response to salt stress. Furthermore, the application of exogenous CTS increased the accumulation of K+ in lettuce but showed no significant effect on the K+/Na+ ratio, as compared with that of plants treated with NaCl alone. These results suggested that exogenous CTS might mitigate the adverse effects of salt stress on plant growth and biomass by modulating the intracellular ion concentration, controlling osmotic adjustment, and increasing antioxidant enzymatic activity in lettuce leaves.

**Keywords:** antioxidant enzymes; chitosan (CTS); lettuce; proline; salinity; soluble sugars

#### **1. Introduction**

Saline stress is a harmful form of abiotic stress that restricts the growth and function of plants and thus can cause a 10%–25% decrease in the yield of many agricultural crops [1]. More than 20% of global farmland is affected by various degrees of salinity, and the farmland area (approximately 20,000 km<sup>2</sup> per year) affected by salinity is increasing each year, which severely limits agricultural productivity [2,3]. Salinity in soils can occur naturally or as a result of human activities. Weathering of rock minerals and flooding by seawater causes inherent soil salinity. Irrigation water with a high salt concentration, excessive chemical fertilization, and poor soil management are the main reasons for an increase in the area of saline–alkali land [4,5]. In some semi-arid and arid areas (e.g., Sahara in North Africa, Saudi Arabia, large parts of Iran and Iraq, parts of Asia, California in the USA, South Africa, and most of Australia), high temperatures and uneven distribution of rainfall result in higher evapotranspiration rates than the size of the leaching fraction, which causes an accumulation of soluble salts in the plough layer [6]. To increase the output of salinized agricultural land, the salt tolerance of plants must be increased and the conditions of saline–alkali land must be improved.

Salt stress negatively influences several processes in plant growth and production by causing ion toxicity, hyperosmotic stress, nutritional imbalance, oxidative damage,

**Citation:** Zhang, G.; Wang, Y.; Wu, K.; Zhang, Q.; Feng, Y.; Miao, Y.; Yan, Z. Exogenous Application of Chitosan Alleviate Salinity Stress in Lettuce (*Lactuca sativa* L.). *Horticulturae* **2021**, *7*, 342. https:// doi.org/10.3390/horticulturae7100342

Academic Editors: Agnieszka Hanaka, Jolanta Jaroszuk-Sciseł and ´ Małgorzata Majewska

Received: 24 August 2021 Accepted: 22 September 2021 Published: 24 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

metabolic disorders, and photoinhibition [4,7]. Because of their sessile nature, plants must evolve several mechanisms to adapt to high-salinity environments. The various physiological and biochemical processes that plants use to adapt to salt stress can be grouped into three categories: Osmotic stress, ionic stress, and detoxification responses [8]. Plants' primary response to salt stress is osmotic stress. Plants adjust their osmotic balance by accumulating organic and inorganic osmolytes, such as proline, glycine betaine, soluble sugar, soluble protein, and sodium (Na+) and potassium (K+) ions, to maintain cell turgor [2,9,10]. Na+ and chloride (Cl−) enter the root system through nonselective cation channels, K+ transporters, and Cl<sup>−</sup> transporters [11]. The excessive accumulation of Na+ and Cl− in plant cells and tissues can adversely affect the growth and development of plants by disturbing the water structure, inhibiting enzymes, and creating nutritional imbalance [12,13]. Plants usually maintain a balanced cytosolic Na+/K+ ratio through Na+ and K<sup>+</sup> transporters and channels [12]. Salt stress also causes the accumulation of reactive oxygen species (ROS), which results in oxidative-stress-induced toxic effects in plants. ROS sources, such as superoxide radical (O2 −), hydrogen peroxide (H2O2), and hydroxyl radical (·OH), are generated by plants' photosynthetic and respiratory electron transport chains, xanthine oxidase, and nicotinamide adenine dinucleotide phosphate oxidase [14]. Normally, cellular ROS levels are regulated by enzymatic (e.g., ROS scavenging enzymes) and nonenzymatic scavengers (e.g., ascorbic acid [AsA], glutathione, and carotenoids) to mitigate the ROS-induced damage caused by salt stress [15,16]. However, although plants adopt these strategies to reduce the harmful effect of salt stress, survival in a salty environment is difficult, let alone producing a good yield.

Chitosan (poly[1,4]-2-amino-2-deoxy-D-glucose; CTS) is a biopolymer obtained through the deacetylation of nontoxic and biofunctional chitin from the exoskeleton of crustaceans [17]. Chitosan has three types of functional groups on its backbone: The amino/acetamido group, and primary and secondary hydroxyl groups, which enhance its affinity for ions and various pollutants [18]. CTS is a natural, low-toxicity, biodegradable, environmentally friendly, renewable, and inexpensive resource and has many applications in the agriculture sector [19]. Since the discovery of CTS by Rouget in 1859 [20], several studies have proven its role in enhancing plant growth and increasing plants' abiotic stress tolerance [21], including rice [19], maize [22], safflower and sunflower [23], and creeping bentgrass [24]. The beneficial role of CTS in stress mitigation is broadly attributable to the alleviation of oxidative stress [25] and the increase in water use efficiency [26], mineral nutrient uptake [27], chlorophyll (Chl) content, and photosynthesis [28] caused by CTS. The application of exogenous CTS increases plants' tolerance to several forms of stress, such as drought, salt, osmotic, and low-temperature stress [19,22,23,29]. Certain concentrations of exogenous CTS have been used to increase plants' resistance to several biotic and abiotic stresses by increasing water use efficiency, enhancing antioxidant activity, and regulating the content of osmotic regulation substances and defense gene expression [17,26,30–32].

Lettuce (*Lactuca sativa* L.) is a leafy vegetable mainly consumed raw and in salad mixes [33]. The production and cultivation area of lettuce has increased because of its marketability, sensory characteristics, and health-promoting properties [5,34]. According to the last available FAO data (http://www.fao.org/faostat/en/#data/QC, accessed on 19 September 2021), the global cultivation area and yield of lettuce were 243.97 thousand hectares and 16.31 million tonnes in 2019, an increase of 0.54% and 2.3% over 2018, respectively. The majority of lettuce comes from China, the United States, and India—the world's top three lettuce producers. Lettuce is a moderately to highly salt-sensitive vegetable [35]. Salinity reduces the seed germination rate, leaf number, photosynthesis, and cell growth and increases the accumulation of ROS, which negatively affects lettuce growth and yield [36,37]. Although the negative effects of salinity on lettuce have been studied [36,38], information regarding the effects of CTS on lettuce growth and production under saline conditions is lacking. Therefore, the present study evaluated the effectiveness of exogenous CTS in mitigating the adverse effects of salinity on the growth and physiological attributes of lettuce plants. In addition, this study identified the effects of exogenous CTS on the

accumulation of osmolytes, the biosynthesis of antioxidants, and the activity of antioxidant enzymes in lettuce under saline conditions.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Treatments*

Romaine lettuce (*Lactuca sativa* var. *longifolia* L. cv. Romana, Takii seed, Japan) was used as the test material, and an experiment was conducted from November 2020 to January 2021. Lettuce seeds were sown in urethane cubes (2.3 × 2.3 × 2.7 cm3), and the seedlings were cultivated in a 22 ◦C growth chamber at 200 ± <sup>10</sup> <sup>μ</sup>mol·m−2·s−<sup>1</sup> photosynthetic photon flux for 12 h by using cool white fluorescent lamps (Figure 1A). At 21 days after sowing (DAS), uniform seedlings were selected and transplanted into a cultivation room of the Engineering and Technical Center for Modern Horticulture in Jurong. The plants were grown in a deep-flow hydroponic system in an Enshi formula nutrient solution (electrical conductivity [EC]: 1.5 ± 0.2, pH: 6.9 ± 0.2) [39]. Air pumps were used to oxygenate the nutrient solution and supply a constant stream of oxygen. The lighting for plant cultivation was provided by light-emitting diode lights (Figure 1B). The photosynthetic photon flux density of the light was 200 ± <sup>10</sup> <sup>μ</sup>mol·m−2·s−1, and its photoperiod was 16 h. The air temperature was maintained as 25 ◦C during the day and 20 ◦C at night, with the relative humidity being maintained as 65% ± 5%.

**Figure 1.** The relative spectral photon flux of (**A**) cool white fluorescent lamps and (**B**) LED lights. The wavelengths of light sources were recorded at 380–800 nm with a spectrometer.

All treatments were performed at 7 days after transplanting (28 DAS) into the cultivation room, so that the lettuce seedlings were well adapted to the environment of the cultivation room. The treatments were divided into four groups: (1) The control group, in which the plants were grown in a nutrient solution with water sprayed on the leaf surface; (2) the CTS group, in which the plants were grown under the same conditions as those of the control group and 100 mg/L of CTS (instead of water) was sprayed on the leaf surface; (3) the NaCl group, in which the plants were grown in a nutrient solution containing 100 mM NaCl and water was sprayed on the leaf surface; and (4) the NaCl + CTS group, in which the plants were grown under the same conditions as those of the NaCl group and 100 mg/L of CTS (instead of water) was sprayed on the leaf surface. CTS or water was sprayed on the leaves of the hydroponically grown lettuce continuously for 5 days from

28 DAS. Approximately 30 mL of CTS or water solution was sprayed on both adaxial and abaxial surfaces of leaves for each lettuce plant. For groups (3) and (4), NaCl was dissolved into the nutrient solution after the 5-day CTS induction treatment. Three replications were performed for each treatment, and each replication comprised six plants. Plants of each treatment were sampled at 28 and 49 DAS for further examinations.

#### *2.2. Plant Growth Analysis*

At 28 and 49 DAS, the plants were sampled to determine total leaf area, shoot and root fresh weight (FW), and shoot and root dry weight (DW). Total leaf area was determined using a Li-3100 leaf area meter (Li-Cor, Lincoln, NE, United States). Shoot and root DW was obtained after the plant tissues were dried at 80 ◦C to a constant weight. Chl was extracted in *N,N*-dimethylformamide from fresh lettuce leaves, and the Chl content was determined spectrophotometrically according to the method of Porra et al. [40].

Growth analysis parameters, namely the relative growth rate (RGR), the net assimilation rate (NAR), and the leaf area ratio (LAR), were estimated using the method of Ohtake et al. [41] by using the following equations:

$$\text{RGR} = (1/\text{W})(\Delta \text{W}/\Delta t) = [\ln(\mathcal{W}\_2) - \ln(\mathcal{W}\_1)]/(t\_2 - t\_1),\tag{1}$$

where *W*<sup>1</sup> and *W*<sup>2</sup> are the total DWs of a plant at times *t*<sup>1</sup> (28 DAS) and *t*<sup>2</sup> (49 DAS).

$$\text{VARR} = (1/L)(\Delta \text{W}/\Delta t) = [(\mathbb{W}\_2 - \mathbb{W}\_1)/(t\_2 - t\_1)] \times [\ln(L\_2) - \ln(L\_1)]/(L\_2 - L\_1), \tag{2}$$

where *L*<sup>1</sup> and *L*<sup>2</sup> are the total leaf areas of a plant at times *t*<sup>1</sup> and *t*2.

$$\text{LAR} = L/\text{W} = (L\_1/\text{W}\_1 + L\_2/\text{W}\_2)/2 \tag{3}$$

#### *2.3. Estimation of Leaf Relative Water Content and Electrolyte Leakage*

Relative water content (RWC) was measured using the method of Yamasaki and Dillenburg [42] by adopting the following equation:

$$\text{RWC } (\%) = (\text{FW} - \text{DW}) / (\text{turgrid weight} - \text{DW}) \times 100\tag{4}$$

Electrolyte leakage (EL) was measured using the method described by Ahmad et al. [43]. Leaf disks with a diameter of 13 mm were produced from the leaves in each treatment group and submerged in deionized water to measure ECa. Then, tubes containing the leaf disks were incubated in a water bath at 50–60 ◦C for 25 min to determine the ECb value for each treatment. Finally, the tubes with the leaf disks were boiled at 100 ◦C for 10 min to measure ECc. The EL was calculated using the following equation:

$$\text{EL (\%)}=(\text{EC}\_{\text{b}}-\text{EC}\_{\text{a}})/\text{EC}\_{\text{c}}\times100\tag{5}$$

#### *2.4. Determination of the Potassium and Sodium Contents in Lettuce Leaves*

The potassium and sodium contents in lettuce leaves were determined through inductively coupled plasma optical emission spectrometry (Thermo Fisher Scientific, Cambridge, United Kingdom) by using the method of Zhang et al. [44].

#### *2.5. Estimation of the Proline, Soluble Sugar, and Ascorbic Acid Contents*

The proline content of the leaf samples was determined according to the method described by Bates et al. [45]. The soluble sugar content of the leaves was measured using the anthrone–sulfuric acid method [46]. The ascorbic acid (AsA) content of the leaves was determined according to the method of Kampfenkel et al. [47].

#### *2.6. Examination of H2O2 Content, O2* − *Generation, and Malondialdehyde Content*

The H2O2 concentration of the leaf samples was determined using the method of Patterson et al. [48]. The O2 − generation was assayed spectrophotometrically by measuring the reduction of nitroblue tetrazolium by using the method of Averina et al. [49]. The malondialdehyde (MDA) concentration of the leaf samples was estimated using the method of Heath et al. [50]. The absorbance of the leaf samples was measured at 450, 532, and 600 nm by using a spectrophotometer.

#### *2.7. Enzyme Assays*

The fresh leaf samples were homogenized in phosphate buffer saline (50 mM, pH 7.8), and the homogenate was centrifuged at 10,000 × *g* and 4 ◦C for 15 min. The supernatants were collected to determine the superoxide dismutase (SOD; EC: 1.15.1.1), peroxidase (POD; EC: 1.11.1.7), and catalase (CAT; EC: 1.11.1.6) activity [51]. Protein content was determined using the method of Bradford [52]. The activity of the enzymes was expressed in units per milligram of protein.

#### *2.8. Statistical Analysis*

The data are presented as the means ± standard errors (SEs) of the three replications for each treatment. Statistical analysis was performed using one-way analysis of variance with Tukey's HSD test (SPSS v. 18.0, IBM Inc., Chicago, IL, USA). *p* values of ≤0.05 were considered significant.

#### **3. Results**

#### *3.1. Exogenous CTS Improved the Growth and Biomass of Lettuce under NaCl Stress*

Compared with the control plants, the lettuce plants exposed to NaCl stress exhibited considerably inhibited plant growth in terms of a lower total leaf area, shoot FW, and shoot DW (Table 1). The total leaf area, shoot FW, root FW, shoot DW, and root DW of the NaCl group were 67.3%, 60.3%, 73.8%, 66.5%, and 51.6% lower, respectively, than those of the control group (Table 1). The application of 100 mg/L of exogenous CTS mitigated the lettuce growth inhibition caused by the salinity stress (Figure 2A). The total leaf area, shoot FW, root FW, shoot DW, and root DW of the NaCl + CTS group were 141.2%, 127.3%, 72.3%, 95.0%, and 60.0% higher, respectively, than those of the NaCl group (Table 1). The total leaf area, shoot FW, root FW, shoot DW, and root DW of the NaCl + CTS group were 21.2%, 40.4%, 31.5%, 34.7%, and 22.6% lower, respectively, than those of the control group (Table 1). However, no significant change in the aforementioned growth parameters was observed between the CTS and control groups (Table 1). The Chl a, Chl b, and total Chl contents of the NaCl group were 14.4%, 20.6%, and 16.1% lower, respectively, than those of the control group (Table 1). The Chl a and total Chl contents of the NaCl + CTS group were 10.1% and 8.6% higher, respectively, than those of the NaCl group (Table 1). The Chl b and total Chl contents of the NaCl + CTS group were 17.3% and 8.9% lower, respectively, than those of the control group (Table 1). In addition, the Chl a content of the plants subjected to exogenous CTS treatment alone were significantly higher than that of the control group (Table 1).

Plant biomass is strongly and positively correlated to RGR, and plant growth analysis decomposes RGR into NAR and LAR. In order to determine how physiological and morphological traits contribute to the plant biomass of each group, the growth analysis parameters of each group were estimated using total plant DW and total leaf area as described in the aforementioned text. The RGR of the NaCl group was significantly lower than that of the control group and corresponded to the lowest DW (Figure 2B, Table 1). The decrease in the RGR of the NaCl group was mitigated by the application of 100 mg/L of exogenous CTS (Figure 2B). A similar trend was observed in NAR, and the lowest NAR was observed for the NaCl group. Moreover, an insignificant difference in NAR was observed between the NaCl + CTS and NaCl groups (Figure 2C). No significant difference in LAR was observed among all the groups (Figure 2D). Similar to the growth parameters, no significant changes in the aforementioned growth analysis parameters were observed between the CTS and control groups.


NaCl + CTS 0.041 ± 0.007 a 29.1 ± 4.5 b 1.56 ± 0.19 b 5.98 ± 0.47 b 0.24 ± 0.03 b 8.18 ± 0.20 b 2.73 ± 0.15 b 10.91 ± 0.35 b Data presented are the means ± SEs (*<sup>n</sup>* = 3). Different letters in each column indicate significant differences (*<sup>p</sup>* < 0.05). CK (control) = 0 mM NaCl + 0 mg/L CTS; CTS = 0 mM NaCl + 100

mg/L CTS; NaCl = 100 mM NaCl + 0 mg/L CTS; NaCl + CTS = 100 mM NaCl +100 mg/L CTS. FW, fresh weight; DW, dry weight; Chl, chlorophyll.

**Figure 2.** Effects of exogenous chitosan (CTS) on (**A**) plant morphology and (**B**–**D**) plant growth analysis parameters of lettuce plants under salt stress. Data presented are the means ± SEs (*n* = 3). Different letters on top of bars indicate a significant difference (*p* < 0.05) according to Tukey's HSD test. CK (control) = 0 mM NaCl + 0 mg/L CTS; CTS = 0 mM NaCl + 100 mg/L CTS; NaCl = 100 mM NaCl + 0 mg/L CTS; NaCl + CTS = 100 mM NaCl +100 mg/L. RGR, relative growth rate; NAR, net assimilation rate; LAR, leaf area ratio.

#### *3.2. Effects of NaCl and CTS on the RWC, EL, and the Contents of Potassium and Sodium of the Lettuce Leaves*

The leaf RWC of the NaCl group was 15.9% lower than that of the control group (Table 2). This decrease in leaf RWC due to salinity was mitigated by the application of exogenous CTS. The leaf RWC of the NaCl + CTS group was 15.7% higher than that of the NaCl group (Table 2). The leaf EL of the NaCl group was 160.9% higher than that of the control group (Table 2). Moreover, the leaf EL of the NaCl + CTS group was 21.2% lower than that of the NaCl group (Table 2). The leaf RWC and EL of the lettuce plants subjected to the exogenous CTS treatment alone were not significantly different from those of the control group (Table 2).

**Table 2.** Effects of chitosan (CTS) on leaf RWC, EL, and the contents of potassium and sodium in leaves of lettuce plants under salt stress.


Data presented are the means ± SEs (*n* = 3). Different letters in each column indicate significant differences (*p* < 0.05). CK (control) = 0 mM NaCl + 0 mg/L CTS; CTS = 0 mM NaCl + 100 mg/L CTS; NaCl = 100 mM NaCl + 0 mg/L CTS; NaCl + CTS = 100 mM NaCl +100 mg/L CTS. RWC, relative water content; EL, electrolyte leakage; FW, fresh weight; K+, potassium; Na+, sodium.

The potassium content of the NaCl group was 28.4% lower than that of the control group (Table 2). The addition of exogenous CTS to the plants treated with NaCl significantly mitigated the inhibition of potassium accumulation in the lettuce leaves (Table 2). The sodium contents of the NaCl and NaCl + CTS groups were 19.7 and 11.1 times higher, respectively, than that of the control group, whereas the accumulation of sodium was 46.0% lower in the NaCl + CTS group than in the NaCl group (Table 2). The K+/Na+ ratio of the NaCl group was significantly lower than that of the control group, and no significant change in the K+/Na+ ratio was observed between the NaCl and NaCl + CTS groups. Non-significant differences were noted in the potassium sodium contents and the K+/Na+ ratio between the CTS and control groups (Table 2).

#### *3.3. Effects of NaCl and CTS on the Proline Content, MDA Content, O2* − *Generation, H2O2 Content, Soluble Sugar Content, and AsA Content of the Lettuce Leaves*

Proline biosynthesis was triggered by salinity stress, which resulted in the NaCl group having a proline content 2.1 times higher than the control group (Figure 3A). Moreover, the proline content of the NaCl + CTS group was 66.5% higher than that of the NaCl group (Figure 3A). MDA, the product of membrane lipid peroxidation caused by ROS, can be used to evaluate the degree of membrane injury under stress [44]. The MDA content of the NaCl group was 127.6% higher than that of the control group (Figure 3B). Moreover, the MDA content of the NaCl + CTS group was 14.3% lower than that of the NaCl group (Figure 3B). The exposure of the lettuce plants to salinity stress induced the production of ROS in cells. The lettuce plants treated with NaCl generated ROS such as H2O2 and O2 −. The H2O2 content and O2 − of the NaCl group were 3 and 1.8 times higher than those of the control group, respectively (Figure 3C,D). The application of exogenous CTS to the plants treated with NaCl slowed the generation of H2O2 and O2 − (Figure 3C,D). The soluble sugar content of the NaCl group was considerably higher than that of the control group (Figure 3E). The soluble sugar content of the NaCl + CTS group was 40.8% higher than that of the NaCl group (Figure 3E). No significant difference in AsA content was observed among the groups (Figure 3F). No significant change in the proline content, MDA content, H2O2 content, superoxide radical production rate, soluble sugar content, and AsA content was observed between the CTS and control groups (Figure 3).

#### *3.4. Effects of NaCl and CTS on the Antioxidant Enzyme Activity in the Lettuce Leaves*

Figure 4 displays the results regarding the effects of NaCl and CTS on the antioxidant enzyme activity in the lettuce leaves. SOD, POD, and CAT exhibited various responses to the different treatments. SOD activity was consistent among all the groups (Figure 4A). However, the POD and CAT activities of the NaCl group were 43.3% lower and 181.9% higher than those of the control group, respectively (Figure 4B,C). The CTS + NaCl group had considerably higher POD and CAT activities than the NaCl group (Figure 4B,C). No significant change in the SOD, POD, and CAT activities was observed between the CTS and control groups (Figure 4).

**Figure 3.** Effects of exogenous chitosan (CTS) on (**A**) proline content, (**B**) MDA content, (**C**) O2 − generation rate, (**D**) H2O2 content, (**E**) soluble sugar content, and (**F**) AsA content in leaves of lettuce plants under salt stress. Data presented are the means ± SEs (*n* = 3). Different letters on top of bars indicate a significant difference (*p* < 0.05) according to Tukey's HSD test. CK (control) = 0 mM NaCl + 0 mg/L CTS; CTS = 0 mM NaCl + 100 mg/L CTS; NaCl = 100 mM NaCl + 0 mg/L CTS; NaCl + CTS = 100 mM NaCl +100 mg/L. MDA, malondialdehyde; O2 −, superoxide radical; H2O2, hydrogen peroxide; AsA, ascorbic acid.

**Figure 4.** Effects of exogenous chitosan (CTS) on activities of (**A**) SOD, (**B**) POD, and (**C**) CAT in leaves of lettuce plants under salt stress. Data presented are the means ± SEs (*n* = 3). Different letters on top of bars indicate a significant difference (*p* < 0.05) according to Tukey's HSD test. CK (control) = 0 mM NaCl + 0 mg/L CTS; CTS = 0 mM NaCl + 100 mg/L CTS; NaCl = 100 mM NaCl + 0 mg/L CTS; NaCl + CTS = 100 mM NaCl +100 mg/L. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.

#### **4. Discussion**

Limited plant growth and productivity are common responses to salinity stress [5]. The data obtained in this study indicate that salinity adversely affected the growth and biomass of lettuce plants and resulted in a significant decrease in their total leaf area, FW, and DW (Table 1). These results are consistent with those of studies on other crops, including tomatoes [53], peppers [54], and chickpeas [43]. CTS, which is a derivative of chitin, has many applications in the agricultural sector because it regulates plant growth and development and increases plants' resistance to a wide range of abiotic and biotic stresses [19,21,55]. The application of exogenous CTS can mitigate the effects of salt stress on plant growth in many crops, such as ajowan [56], maize [57], and wheat [58]. A similar finding was obtained in this study, which indicates that the application of exogenous CTS alleviated the inhibition of the growth of lettuce caused by saline conditions (Table 1, Figure 2A). Growth analysis is a widely used analytical method for characterizing plant growth [59]. Plant biomass is strongly and positively correlated with RGR. We observed that the RGR of the NaCl group was significantly lower than that of the control group (Figure 2B), which indicates that the lettuce exposed to saline conditions accumulated less biomass than the lettuce grown under normal conditions during the growth stage. The NAR (average growth per unit leaf) of the NaCl group was significantly lower than that of the control group (Figure 2C). However, the LAR of the lettuce was relatively

consistent between the saline and normal conditions (Figure 2D). These results indicate that the decrease in the RGR of the lettuce under salt stress was mainly associated with NAR and less associated with LAR. The exogenous application of CTS markedly increased the RGR of the lettuce under salt stress (Figure 2B), which proves that exogenous CTS can mitigate the inhibition of plant growth caused by salinity. However, we observed only a minor variation in NAR between the NaCl + CTS and NaCl groups (Figure 2C). Thus, the increase in the RGR of the NaCl + CTS group may have been related to both NAR and LAR. The Chl content of leaves is commonly considered a reliable predictor of the health and photosynthesis capacity of plants during growth [60,61]. Chlorophyll degradation under salt stress is usually related to the accumulation of ROS, which causes lipid peroxidation of chloroplast membranes [62]. In this study, the Chl content of the NaCl group was considerably lower than that of the control group (Table 1), which is in agreement with previous findings [63]. This result might be ascribable to the impaired biosynthesis or accelerated degradation of Chl pigment under saline conditions [43,64]. The application of exogenous CTS to the lettuce under saline conditions increased the Chl a and total Chl contents (Table 1). Similarly, Zou et al. [62] found that exogenous polysaccharides increased the Chl a content in wheat seedling leaves under salt stress. Chl a is responsible for the absorption of light and the initiation of photosynthesis. Excessive amounts of salt accumulated in chloroplasts can exert a direct toxic effect on photosynthesis through the destruction of pigment–protein complexes [65]. Thus, the increases in Chl a content in NaCl + CTS group are possibly because exogenous CTS protected Chl a from degradation in salt-stressed lettuce leaves, leading to high efficiency in photosynthesis. Moreover, the NaCl + CTS group did not exhibit a significantly higher Chl b content than the NaCl group possibly because Chl b tends to transform into Chl a when plants are subjected to saline conditions [66].

Leaf RWC is an accurate measure of the status of water in plants and indicates the water content of a leaf relative to the maximum amount of water that the leaf can contain under full turgidity [67]. Our study indicates that salinity decreased leaf RWC substantially; however, the exogenous application of CTS positively affected the leaf RWC of the lettuce exposed to saline conditions (Table 2). Geng et al. [24] also found that exogenous CTS application significantly increased the leaf RWC and water use efficiency in creeping bentgrass under salt stress, which contributes to maintaining a better water status in plants exposed to salinity stress. Thus, the results obtained from the current study were possibly due to the regulation of the balance between the water supply and leaf transpiration in the lettuce of the NaCl + CTS group. Under normal conditions, the Na+ content was very low in lettuce leaves, and no significant change was observed between the CK and CTS groups (Table 2). Salt stress significantly increased Na<sup>+</sup> content but decreased the K+ content in lettuce leaves (Table 2). The application of exogenous CTS increased the K<sup>+</sup> content and decreased the Na<sup>+</sup> content in the leaves of lettuce exposed to saline conditions, but the K<sup>+</sup> content in leaves was still lower than that of the control group (Table 2). A similar trend was also found in salt-stressed wheat seedlings applied with polysaccharides [62]. The accumulation of high levels of Na<sup>+</sup> in plant tissues subjected to saline conditions has a devastating effect on the metabolism of cytoplasm and organelles. As cytosolic Na+ is noxious to cells, so too is chloroplastic Na+ accumulation [12]. Excess Na<sup>+</sup> will cause an imbalance in cellular Na<sup>+</sup> and K <sup>+</sup> homeostasis, which often leads to a low K+/Na+ ratio [68]. Hereafter, plants will suffer from K<sup>+</sup> deficiencies stemming from the competitive inhibition of its uptake by Na<sup>+</sup> in plants exposed to salt stress. Potassium is an essential nutrient for plant growth and production, it is involved in the balance of osmotic pressure and the regulation of stomatal closure, and it affects photosynthesis and enzymatic activity [44]. Maintaining a high shoot K+/Na+ ratio is an important trait of plant salt tolerance [69]. In the current study, the shoot K+/Na+ ratio decreased dramatically under saline conditions due to excessive Na<sup>+</sup> accumulation in leaves. However, K+ accumulation in NaCl + CTS-group plants was accompanied by a higher K+/Na+ ratio but not a significant difference compared to that of the NaCl group. These results, combined with the mitigation of growth inhibition, suggest that exogenous CTS might facilitate plant growth by regulating the nutritional balance and reducing ion toxicity. A previous study has shown that polysaccharides enhanced salt tolerance in wheat by maintaining a high K+/Na+ ratio through regulation of several Na+/K+ transporter genes, coordinating the efflux and compartmentation of Na+ [58]. The study of Geng et al. [24] found that CTS enhanced salt overly sensitive pathways and upregulated the expression of *AsHKT1* and genes encoding Na+/H+ exchangers under saline conditions, thus inhibiting Na+ transport to the photosynthetic tissues. In the present study, although the lower Na<sup>+</sup> content in leaves was observed in the NaCl + CTS group at harvest time, the Na+ levels in shoot and root were not evaluated in a time-response manner, and the gene expressions related to Na+ transport still have to be analyzed. To further understand the CTS-induced salt tolerance in lettuce, future studies need to focus on examining the accumulation pattern of Na+ in shoot and root and investigating the gene expressions involved in Na+ transport.

Osmotic regulation is a major adaptation mechanism for plants to resist salt stress. High levels of osmolytes, such as proline, soluble sugars, and soluble proteins, are accumulated in the cytosol and other organelles to adjust osmotic pressure [17,44]. The high accumulation of proline in the NaCl group indicated the crucial nature of this osmolyte in the osmotic adjustment under saline conditions (Figure 3A). Under salt stress, proline can regulate osmotic potential, stabilize the cellular structure, reduce damage to the photosynthetic apparatus, and induce the expression of salt stress-responsive genes, consequently enhancing the adaption of the plant to saline conditions [58]. The application of exogenous CTS increased the proline level and Chl a content of the lettuce leaves subjected to saline conditions (Figure 3A), which suggests that exogenous CTS not only balances osmosis in cells but also protects their photosynthetic machinery. It had been proved that proline accumulation might be a result of a salt-induced increase in N metabolism [70]. A previous study found in wheat that exogenous CTS could effectively enhance N metabolism [71]. Thus, it is interesting to further study whether CTS increases proline levels by modulating N metabolism in lettuce plants during salt stress. The quantity of another vital osmolyte, namely soluble sugar, increased in the lettuce under saline conditions regardless of whether the leaves were treated with exogenous CTS; however, the soluble sugar levels of lettuce leaves treated with exogenous CTS were considerably higher than those of lettuce leaves treated with NaCl alone (Figure 3E). Because soluble sugar plays a key role in many physiological and biochemical processes, including photosynthesis, ROS scavenging, and the induction of adaptive pathway destructive conditions, a substantial increase in the total soluble sugar content may effectively protect lettuce plants exposed to NaCl stress [72,73].

Salt stress triggers the generation of a large number of ROS, such as O2 −, H2O2, and ·OH, which poses challenges to plant cells [74,75]. The excessive generation of ROS causes the oxidation of lipids and proteins and the breakage of nucleic acids and limits the effectiveness of enzymes, which results in abnormalities at the cellular level and thus the inhibition of plant growth [43,76]. MDA, which is the final product of membrane lipid peroxidation caused by ROS, is generally an indicator of the degree of cell membrane damage in plants subjected to stress [77,78]. The results of our experiment reveal that the O2 −, H2O2, MDA contents, and EL of the NaCl group were considerably higher than those of the control group, which indicates that the integrity and stability of the cell membrane decreased due ROS-induced oxidative damage (Figure 3B–D; Table 2). The O2 −, H2O2, and MDA contents and EL of the CTS + NaCl group were lower than those of the NaCl group (Figure 3B–D; Table 2). These results suggest that CTS can mitigate oxidative damage and regulate the stability of the cell membrane system under saline conditions. Moreover, the observed significant increases in Chl a content and reductions in the MDA level in lettuce leaves of NaCl + CTS group also proved that exogenous CTS reduced lipid peroxidation and mitigated the salt-induced reduction in chlorophyll content (Figure 3B; Table 1). Similarly, Turk [57] reported that the application of exogenous CTS decreases ROS levels and lipid peroxidation in peppers under salt stress.

Plants possess a wide range of radical scavenging systems to manage oxidative damage, including antioxidative enzymes, such as SOD, POD, and CAT, and nonenzymatic compounds, such as proline and AsA [44,79–81]. SOD is a major O2 − scavenger that catalyzes O2 − to H2O2 and O2 [82]. Thereafter, the toxic H2O2 can be removed by POD, CAT, or ascorbate peroxidase [83,84]. In the current study, the O2 −, H2O2, and MDA contents of the NaCl group were considerably higher than those of the control group (Figure 3B–D). Moreover, the CAT and POD activities of the NaCl group were lower and higher, respectively, than those of the control group (Figure 4). The exogenous application of CTS significantly decreased the O2 −, H2O2, and MDA levels and considerably increased the POD and CAT activities (Figure 3B–D and Figure 4). These results are consistent with those of studies on maize [57] and suggest that the exogenous application of CTS can mitigate the damage to the cell membrane system by salt stress by increasing the POD and CAT activities in lettuce leaves. The enzymatic activities of POD and CAT may have played a more crucial role than that of SOD in scavenging the overproduction of ROS in the plants treated with NaCl alone because no significant difference was observed in SOD activity among all the groups (Figure 4). However, the current study did not investigate the expression patterns of *SOD*, *CAT*, and *POD* genes, and additional studies should be conducted on this topic. In addition, studies have indicated that proline and AsA are potent antioxidants that can scavenge various types of ROS and shield the cell from oxidative damage [85,86]. In the current study, the application of exogenous CTS significantly increased the proline content of the lettuce leaves (Figure 3A), indicating that lettuce may accumulate high levels of proline to scavenge ROS, decrease oxidative damage, and safeguard cell membranes from the adverse effects of salt stress. No significant difference was observed in the AsA contents of the CTS + NaCl and NaCl groups (Figure 3F), which suggests that AsA may not play an important role in maintaining the strong antioxidant capacity of lettuce under saline conditions. A current study also found that exogenous oligo-alginate in NaCl-treated plants did not change the AsA content [87].

#### **5. Conclusions**

CTS, a natural polysaccharide, has many applications in the agriculture sector as an exogenous additive substance, being both safe and cheap. In the present study, the effects of exogenous CTS on lettuce plants under salt stress were investigated. The results showed that exogenous CTS could improve plant growth and biomass under salt stress. Exogenous CTS application increased proline and soluble sugar accumulations and enhanced peroxidase and catalase activities, thereby reducing oxidative damage to leaves. The CTS also curbed the accumulation of sodium but enhanced the accumulation of potassium in the leaves of NaCl-treated plants. These outcomes may help optimize the production technology of lettuce under saline conditions. However, the mechanism of CTS on alleviating salinity damage is still not fully understood. Future studies should focus on analyzing Na+/K+ transporter gene expressions and possible signal transduction pathways involved in CTS-regulated increased tolerance of lettuce plants to salt stress. As a biopolymer, the presence of amine and hydroxyl groups in CTS may also prevent Na+ from reaching the photosynthetic tissue by chelating part of it at the root/lower tissue level, which needs further exploration depending on the CTS application method.

**Author Contributions:** Conceptualization, G.Z. and Y.W.; methodology, G.Z., Z.Y. and Y.W.; validation, Q.Z. and Y.F.; data analysis, G.Z., Y.W. and Q.Z.; writing—original draft preparation, G.Z. and Y.M.; writing—review and editing, K.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China, 19KJB210011; Innovation Training Program for College Students of Jiangsu Vocational College of Agriculture and Forestry, 202113103005y.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Cullen Pitney and Randy Johnson for many helpful comments and suggestions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Adaptive Morphophysiological Features of** *Neottia ovata* **(Orchidaceae) Contributing to Its Natural Colonization on Fly Ash Deposits**

**Maria Maleva 1,\*,†, Galina Borisova 1,†, Nadezhda Chukina 1, Olga Sinenko 1, Elena Filimonova 2, Natalia Lukina <sup>2</sup> and Margarita Glazyrina <sup>2</sup>**


**Abstract:** In previous decades, some species of the Orchidaceae family have been found growing in man-made habitats. *Neottia ovata* is one of the most widespread orchids in Europe, however it is quite rare in Russia and is included in several regional Red Data Books. The purpose of this study was to compare the chemical composition and morphophysiological parameters of *N. ovata* from two forest communities of the Middle Urals, Russia: natural and transformed (fly ash dump of Verkhnetagil'skaya Thermal Power Station) for determining orchid adaptive features. The content of most of the studied metals in the underground parts (rhizome + roots) of *N. ovata* was considerably higher than in the leaves, which diminished the harmful effect of toxic metals on the aboveground organs. The adaptive changes in the leaf mesostructure of *N. ovata* such as an increase in epidermis thickness, the number of chloroplasts in the cell, and the internal assimilating surface were found for the first time. The orchids from the fly ash deposits were characterized by a higher content of chlorophyll *b* and carotenoids than plants from the natural forest community that evidenced the compensatory response on the decrease in chlorophyll *a*. The ability of *N. ovata* from the transformed habitat to maintain a relatively favorable water balance and stable assimilation indexes further contribute to its high viability. The study of orchid adaptive responses to unfavorable factors is necessary for their successful naturalization and introduction into a new environment.

**Keywords:** orchid; transformed ecosystems; fly ash; metals; adaptive responses; water exchange; leaf mesostructure; photosynthetic pigments; photosynthesis; plant introduction

#### **Highlights**

*Neottia ovata* successfully colonize the fly ash dump (FAD) due to less phytocoenotic stress. *N. ovata* plants from transformed habitat demonstrate high viability.

Sequestration of metals mainly in underground organs reduced harmful effect on orchid plants.

Natural orchid colonization of FAD was facilitated by adaptive structural and functional changes.

The FAD plants were characterized by the higher chlorophyll *b* and carotenoids content. *N. ovata* from FAD maintained a relatively favorable water balance and stable assimilation indexes.

#### **1. Introduction**

The Orchidaceae family has a broad variety of more than 28,000 species distributed in about 763 genera and widespread from the Arctic tundra to tropical Brazilian rainforests [1,2]. It includes species with complex adaptations to pollination by specific insect

**Citation:** Maleva, M.; Borisova, G.; Chukina, N.; Sinenko, O.; Filimonova, E.; Lukina, N.; Glazyrina, M. Adaptive Morphophysiological Features of *Neottia ovata* (Orchidaceae) Contributing to Its Natural Colonization on Fly Ash Deposits. *Horticulturae* **2021**, *7*, 109. https://doi.org/10.3390/ horticulturae7050109

Academic Editors: Jolanta Jaroszuk-Sciseł, Małgorzata ´ Majewska and Agnieszka Hanaka

Received: 7 April 2021 Accepted: 4 May 2021 Published: 11 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

species and very different life strategies: from epiphytic to terrestrial, from evergreen to completely chlorophyll-free [3,4]. Studies have been conducted on orchids' taxonomy, morphology, ecology, breeding [5–7], pollination [8–10], genetics [11,12], mycorrhizal association [13–15], etc. At the same time, the physiological parameters of Orchidaceae species are still less studied and need much more attention [16].

Changing natural habitats have caused the extinction of many orchid species [17]. However, some orchids, especially in temperate regions of Europe and North America, have been found in anthropogenically disturbed territories, such as industrial dumps formed after the excavation and extraction of coal, iron, and some trace elements, and the fly ash dumps of thermal power plants [13,14,18–21].

The monitoring of vegetation restoration on disturbed lands in the Middle Urals, Russia, has shown that dumps from mining and processing industries are often colonized by some rare orchid species at the initial stages of the forest phytocoenoses formation [22–24]. Low competition in man-made habitats contributes to the conservation of the gene pool of Orchidaceae species. The local populations of *Neottia ovata* (L.) Bluff and Fingerh. (syn. *Listera ovata* (L.) R. Br. or Common twayblade) are of particular interest, as they have been found in recent years in disturbed territories of the Middle Urals, including fly ash dumps [22].

Common twayblade is one of the widespread orchids in Europe, especially in the British Isles [25]. However, this species is quite seldom encountered in Russia and has the status of a "rare species" in many regional Red Data Books, including the Red Book of Sverdlovsk Region [26]. This is a short-rhizome herbaceous perennial, mesophyte, European–West Asian, boreal-immoral species [3,25,27]. Like other orchids, *N. ovata* is characterized by low competitiveness. *N. ovata* grow on both fertile and poor soils. Sometimes it is found in disturbed habitats, along roadsides and railways, and in abandoned limestone quarries [4]. The *N. ovata* is a typical entomophile, the spectrum of pollinators is very wide [9]. This species reproduces well both by seeds and vegetatively [28]. For the germination of seeds in the first years of life, the presence of mycorrhiza is necessary [4].

Fly ash is considered a problematic form of solid waste throughout the world [29,30]. It is well known that fly ash substrates are characterized by low microbiological activity, an insufficient supply of nutrients, especially nitrogen, and adverse physicochemical properties [31]. Moreover, fly ash may also contain toxic concentrations of As, Cd, Cr, Pb, Co, Cu, etc. [29–31].

Different plant species growing in stressful environments show great variation in their tolerance mechanisms [29]. Unfavorable abiotic factors affect photosynthesis, respiration, water regime, and mineral nutrition, leading to impaired growth and development [21–24]. Photosynthesis is the main fundamental process that determines the productivity of plants [32,33]. The leaves are the primary photosynthetic organs, serving as key sites where the absorption of light and CO2 assimilation take place. The internal organization of the leaf is influenced by environmental factors such as the chemical properties of soil substrates, light, temperature, and water availability. Leaf structure is known to be highly plastic in response to growing conditions, varying greatly in morphology, anatomy, and physiology [32–34]. The investigation of plant leaf traits and its responses to environmental change has increasingly gained more attention in recent decades [34–37]. Maintaining the functional activity of the photosynthetic apparatus in stressful conditions is one of the essential prerequisites that allow plants to colonize transformed territories.

The purpose of this study was a comparative analysis of the chemical composition and the structural and functional parameters of *N. ovata* from natural and transformed (fly ash dump) habitats. This will allow us to identify the adaptive morphophysiological characteristics of this species that contribute to its colonization on infertile technogenic substrates.

#### **2. Materials and Methods**

#### *2.1. Study Area*

The research was conducted in the Middle Urals, Russia (subzone of the southern taiga). The southern taiga subzone is characterized as moderately cold in terms of heat supply and over-humidified in terms of moisture availability. The average annual temperature is 1.9 ◦C, the annual precipitation is almost 570 mm, and the hydrothermal coefficient is about 1.5 [32]. The fly ash dump formed by brown coal ash is located on an area of 1.25 km2. The fly ash deposits formed after mining (1968–1970) were left for colonization by natural forest [22,31,38]. The investigation was carried out in the vicinity of Verkhniy Tagil town (Sverdlovsk region) during the period of orchid blooming (mid-July 2018–2019). All samples were collected during the same period (from 15 to 18 July) under similar weather conditions (temperature during the daytime was 23 ± 3 ◦C and the relative humidity was about 60%). Two naturally growing orchid populations were studied: P-1 (57◦20 13" N 60◦01 43" E) from the natural forest community (NFC) near Belorechka village and P-2 (57◦20 45" N 59◦56 46" E) from the fly ash dump (FAD) near Verkhnetagil'skaya Thermal Power Station, VTTPS (Figures 1 and 2). The studied area of each site was about 400 m2.

**Figure 1.** Map of Sverdlovsk region, Russia. Detailed presentation of the locations of studied *N. ovata* populations: P-1—from natural forest community near Belorechka village; P-2—from the fly ash dump near Verkhnetagil'skaya Thermal Power Station (VTTPS).

The natural forest community was represented by a mixed forest. The soil of this site was is sod-podzolic. The age of trees was between 80 and 100 years and the tree crown density was between 0.5 and 0.6. The height of the first layer of trees was 10–24 m and the second layer was 6–12 m. Coniferous species (*Picea obovata* Ledeb., *Larix sibirica* Ledeb., *Pinus sylvestris* L.) predominated in this forest. *N. ovata* individuals in P-1 grew mainly under the deciduous species *Betula pendula* Roth and *B. pubescens* Ehrh., as well as in small glades and meadows. The total projective shrub cover was 30–50% and contained *Tilia cordata* Mill., *Sorbus aucuparia* L., *Padus avium* Mill., *Rosa majalis* Herm., *R. acicularis* Lindl., and *Rubus idaeus* L. The total projective herbaceous cover was 70–80%, which reached up to 100% in the glades. Dominant among the herbaceous species were

*Calamagrostis arundinacea* (L.) Roth, *Aegopodium podagrária* L., *Círsium heterophyllum* (L.) Hill., *Brachypodium pinnatum* (L.) Beauv., *Geranium sylvaticum* L., *Lathyrus vernus* (L.) Bernh., *Anthoxanthum odoratum* L., *Alchemilla vulgaris* L., *Poa pratensis* L., *Melica nutans* L., *Vicia sepium* L., and *Ranunculus acris* L. The moss–lichen layer was weak. In total, more than 90 species grew on the investigated site and the species richness was 31 species per 100 m2.

**Figure 2.** Flowering *N. ovata* plants from: (**a**) natural forest community (P-1) and (**b**) fly ash dump (P-2); (**c**) naturally colonized fly ash dump of VTTPS; *N. ovata* (**d**) inflorescence and (**e**) flower.

The orchid population P-2 was found in the young forest community formed during the natural revegetation of the fly ash deposits. Soil formation was proceeding according to zonal type in the fly ash substrate under the forest communities [38]. The tree crown density was 0.4, which reached up to 0.6 in some places. The 35–40-year-old forest community was dominated by *B. pendula*, *Populus tremula* L., *P. sylvestris*, *P. obovata*, and, less often, *B. pubescens*. In the undergrowth, there were singular instances of *P. obovata*, L. sibirica, and *Abies sibirica* Ledeb. The total projective shrub cover was 10–30% and contained *Salix myrsinifolia* Sm., *Sorbus aucuparia* L., *Padus avium* Mill., *Viburnum opulus* L., and *Chamaecytisus ruthenicus* (Fisch. ex Wołoszcz.) Klásková. The total projective herbaceous cover was 20–25%, in some places reaching 70%. The dominant species were *Platanthera bifolia* (L.) Rich., *Calamagrostis epigejos* (L.) Roth, *Amoria repens* (L.) C. Presl, *Pyrola rotundifolia* L., *Orthilia secunda* (L.) House, *Poa pratensis* L., *Festuca rubra* L., and *Equisetum arvense* L. The moss–lichen layer was weak. In total, more than 60 species grew on this site and the species richness was 19 species per 100 m2.

#### *2.2. Plant and Soil Substrate Sample Collection, Preparation and Analysis*

From each site, no more than 20% of the total number of orchids in the studied populations were randomly selected to minimize the damage to these populations. Since the populations differed in terms of the number of individuals (39 and 194 in P-1 and P-2, respectively), four flowering orchid plants from natural forest population (P-1), and eight from the fly ash deposits (P-2), were carefully excavated with part of the soil substrate to preserve the underground organs (rhizome and roots). The studied plants from both the

sites were in the range of 50–60 cm in length. The samples were placed into separate sterile 10 L bags to minimize dehydration and transferred to the laboratory for further analysis.

The soil substrate samples (about 2 kg) were taken from each orchid root zone at a depth of 0–15 cm, and a composite sample was formed for analysis. Subsequently, the soil samples were air dried, homogenized, passed through a sieve (<2 mm), and preserved for physicochemical analysis (pH, electrical conductivity, total dissolved solids, total and available metal concentrations).

Before chemical analysis, the plants were carefully washed by ultrasonication (UM-4, Unitra Unima, Olsztyn, Poland) and finally with deionized water (Milli-Q system, Millipore SAS, Molsheim, France). The leaves, rhizome, and roots from each individual plant were separated and then dried for 24 h at 75 ◦C along with soil samples. Afterwards, the dried samples were weighed and digested with concentrated HNO3 (analytical grade) using a MARS 5 Digestion Microwave System (CEM, Matthews, NC, USA). The available form of metals was analyzed by extracting the soil sample (5 g) with 10 mL of 0.5 M nitric acid solution as described earlier [24]. All the samples were prepared using deionized Millipore water. The concentrations of K, Ca, Mg, Fe, Zn, Mn, Pb, Cu, Ni, Cr, and Co in all the samples were determined using an atomic absorption spectrometer AA240FS (Varian Australia Pty Ltd., Mulgrave, Victoria, Australia) [24]. Standard reference materials (JSC Ural Chemical Reagents Plant, Verkhnyaya Pishma, Russia) were used for the preparation and calibration of each analytical batch. The calibration coefficients were maintained at a high level of no less than 0.99.

The bioconcentration factor (BCF) was calculated as the ratio of the metal concentration in the underground/aboveground organs to the available concentration in the soil. The translocation factor (TF) was calculated as the ratio of metal concentration in the leaves to the concentration in the rhizome + roots.

The pH, electrical conductivity (EC), and total dissolved solids (TDS) of the soil–water suspensions (1:2.5; *w/v*) were measured using a portable multivariable analyzer HI98129 Combo (Hanna Instruments GmbH, Graz, Austria). The total nitrogen and phosphorus content in the *N. ovata* leaves and rhizome + roots were measured spectrophotometrically at 440 and 640 nm, respectively, after wet digestion with an acid mixture of HClO4 and H2SO4 (1:10; *v/v*). The total nitrogen was measured after the reaction with Nessler's reagent [39], whereas the total phosphorus was determined by standard method using ammonium molybdate in the acid medium [40].

#### *2.3. Morphological, Anatomical Parameters and Mycorrhiza Assay*

Twenty flowering plants from each population were used to study shoot and inflorescence length, the number of flowers, and the total leaf area under in situ conditions. To calculate the leaf area, each leaf was photographed on graph paper and digital image analysis was performed using special MesoPlant software (OOO SIAMS, Ekaterinburg, Russia). From the lower leaf of 10 plants about 30 leaf discs (0.7 cm diameter) were fixed in 3.5% glutaraldehyde solution in a phosphate buffer (pH 7.0, *v/v*) in order to study the mesostructural parameters: leaf mesophyll and epidermis thickness (μm), number of cells per unit leaf area (thousand cm−2), chloroplasts per mesophyll cell (pieces), and cell and chloroplast volumes (μm3). The transverse sections of the leaf discs were obtained using a freezing microtome MZ-2 (JSC Kharkov plant "Tochmedpribor", Kharkov, Ukraine). All measurements were carried out in 30 replicates using a Meiji MT 4300 L light microscope (Meiji Techno, San Jose, CA, USA). The quantitative parameters of the mesophyll were determined with a computer-assisted protocol based on MesoPlant software (OOO SIAMS, Ekaterinburg, Russia). The number of cells per unit of leaf surface area was counted in a Goryaev cytometer after tissue maceration in a 20% KOH solution (*v*/*v*) with heating at 80–90 ◦C. All other measurements were carried out on leaf discs preliminarily macerated with 5% chromic acid dissolved in 1 N HCl (*v/v*) [34].

The quantitative indices of the leaf mesophyll were determined according to Mokronosov [31], modified by Ivanova and P'yankov [34]. The cell volume per chloroplast (CVC, μm3) was calculated as the ratio of cell volume to the number of chloroplasts per cell. The chloroplast membrane index (CMI, cm<sup>2</sup> cm−2) was calculated as the ratio of the total surface area of the outer membranes of chloroplasts to the unit of leaf surface area [35].

Fresh roots of *N. ovata* from both the studied sites were used for investigating mycorrhizal association. Root tips up to 1.5 cm were cut into 20 μm cross sections with a freezing microtome and analyzed under a Meiji MT 4300 L light microscope (Meiji Techno, San Jose, CA, USA) [24].

#### *2.4. Physiological and Biochemical Parameters Assay*

To measure photosynthesis and transpiration, freshly dug-up plants with bulk soil (as described earlier in Section 2.2) were transported to the laboratory and studied no later than 3 h after collection to minimize the dehydration.

The gas exchange (μM CO2 m−<sup>2</sup> s−1) and transpiration rate (mM H2O m−<sup>2</sup> s−1) were measured with the lower leaf of four plants using a LI-6400XT portable infrared gas analyzer (LI-COR, Lincoln, NE, USA) with a LED Light Source chamber (3 × 4 cm). The following parameters were set: operating at an ambient concentration of CO2 and humidity, the temperature was +23 ◦C and the saturating light intensity of 1600 μM m−<sup>2</sup> s−1. This value of light intensity was experimentally established earlier by constructing average light curves. The CO2 uptake was recalculated to mg of CO2 per unit leaf area (dm2), per mg of chlorophyll (Chl *a+b*), and per chloroplast (108) per hour; the transpiration rate was recalculated to g of H2O per unit leaf area (dm2) per hour. Subsequently, fresh leaf cuttings (0.7 cm diameter) from these leaves were used to measure the water exchange and photosynthetic pigment content.

The relative water content (RWC, %) and water saturation deficit (WSD, %) of the plant tissue were measured by floating disc method and calculated according to Hellmuth [41]. The fresh leaf cutting was immediately weighed to obtain FW and then saturated by submerging the sample in distilled water for 3 h. Afterwards, the surface water was blotted carefully, the discs were weighed to obtain the saturated weight, and later dried 24 h at 75 ◦C to determine the dry weight. The fresh weight (FW) to dry weight (DW) ratios were used for further calculations. Simultaneously, the part of the leaf discs was immediately used for photosynthetic pigment determination. Three discs from each plant (about 40–50 mg of FW) were homogenized in 2 mL of a cold 80% acetone solution (*v/v*) with addition of a small amount of CaCO3 to protect the pigments from oxidation, and centrifuged at 8000× *g* for 10 min. The homogenate was decanted, acetone solution was added to the precipitate and stirred again; this procedure was repeated threefold until the precipitate was completely discolored. The content of chlorophyll *a* (Chl *a*), chlorophyll *b* (Chl *b*), and carotenoids (CAR) was determined spectrophotometrically ("APEL" PD-303 UV) at wavelengths of 470, 647, and 663 nm, respectively, and calculated according to Lichtenthaler [42], and expressed as mg g−<sup>1</sup> DW. The physiological and biochemical parameters were determined in four biological and three analytical replicates.

#### *2.5. Statistical Analysis*

The values are presented as mean values of 5 replicates for the physicochemical analysis of the soil and the elemental analysis of plant samples, 30 replicates for structural characteristics, and 12 replicates for physiological and biochemical plant parameters with standard error (SE). After checking the normality by Shapiro–Wilk test and the homogeneity of variance by Levene's test, the differences between the studied orchid populations were determined with the nonparametric Mann–Whitney *U*-test, *p* < 0.05. The relationship between different parameters was determined by Spearman's rank correlation coefficient. Asterisks in the tables and figures indicate significant differences between the studied populations.

#### **3. Results**

#### *3.1. Brief Description of Studied Populations*

In NFC, *N. ovata* plants were found both as single individuals and in groups of up to three individuals. The total number of *N. ovata* plants in P-1 was 39, with a density of 0.10 individuals per 1 m2; the age spectrum was dominated by flowering plants (71%).

The distribution of *N. ovata* plants in P-2 was uneven. The orchid population from FAD was young and vegetatively oriented, with pregenerative individuals predominating (78%). The total number of *N. ovata* plants in P-2 was 194 individuals and the density was 0.49 individuals per 1 m2.

Orchid mycorrhiza, represented by pelotons, were found in the root cells of *N. ovata* from both studied populations. The intensity of mycorrhizal association was high and ranged from 96 to 98%.

#### *3.2. Soil and Plant Composition*

3.2.1. Physicochemical Characteristics and Metal Content in Soil Substrates

The pH of the soil substrates varied between acidic and slightly acidic for both sites. At the same time, the pH of the fly ash substrate was slightly higher than that of the natural forest soil (Table 1). The EC and TDS values in the soil substrate from NFC were higher (1.7-fold on average) than in FAD.

**Table 1.** The pH (water solution), electrical conductivity and total dissolved solids in soil substrates (0–15 cm depth) from the natural forest community (NFC) and the fly ash dump (FAD).


<sup>1</sup> Data is presented as mean ± SE (*<sup>n</sup>* = 5); <sup>2</sup> In the brackets are the minimum and maximum values. Asterisks (\*) indicate significant differences between the studied habitats according to Mann–Whitney *U-*test (*p* < 0.05).

The total metal contents in the NFC soil were found in the following order: Fe > Ca > Mg > Mn > K > Zn > Pb > Cu > Cr > Ni > Co (Table 2). A similar trend was noted in the metal content distribution in the FAD substrate, with the exception of Mn and K that switched places. The largest difference between the two sites was found for Mn; its total and available content in the NFC soil exceeded its concentration in the FAD substrate by 3.3 times on average. The total contents of Ca, Mg, Zn, and Cu were also higher in the soil of the natural habitat than in the disturbed one, but the differences between the sites were less noticeable (1.3–2.0 times). Whereas, for K, Pb, and Cr, their content (both total and available) was higher in the FAD substrate (on average by 1.4 times). At the same time, there were no significant differences between the total Fe, Ni, and Co contents in both sites. The available concentration of most of the studied metals (namely, Ca, Mg, Fe, Zn, Mn, Cu, and Ni) in the NFC soil was higher compared to the FAD substrate (on average 2.0 times, Table 2). As for Co, there were no significant differences in the content between the sites studied.


**Table 2.** Total and available metal content in the soil substrates (0–15 cm depth) from the natural forest community (NFC) and the fly ash dump (FAD).

<sup>1</sup> Data is presented as mean ± SE (*<sup>n</sup>* = 5); <sup>2</sup> In the brackets are the minimum and maximum values. Asterisks (\*) indicate significant differences between studied habitats according to Mann–Whitney *U-*test (*p* < 0.05).

#### 3.2.2. Macronutrient and Metal Content in *N. ovata*

The leaves of *N. ovata* from P-2 contained a smaller amount of total nitrogen, while the content of total phosphorus was higher than in the P-1 plants (by 1.2 times, Table 3).


**Table 3.** Macronutrient content in the aboveground and underground organs of *N. ovata* from the natural forest community (P-1) and the fly ash dump (P-2).

<sup>1</sup> Data is presented as mean ± SE (*<sup>n</sup>* = 5). Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

As for content of these nutrients in the *N. ovata* rhizome + roots, there were no significant differences in the nitrogen content between the studied orchid populations, although lower phosphorus content was noted in the plants growing on the fly ash substrate.

As expected, among the studied metals the *N. ovata* plants accumulated K, Ca, and Mg in the greatest amounts (Table 3). The potassium content in the both the aboveground and underground organs of *N. ovata* was 1.4 times higher than in the plants from the FAD while the studied populations did not significantly differ in terms of Ca and Mg content.

As for the other metals, they accumulated to the greatest extent in the *N. ovata* underground organs (Figure 3). The content of toxic elements such as Pb and Cr was higher in the rhizome + roots of the plants colonizing FAD (by 43 and 26%, respectively). In contrast, the content of essential Cu and Fe was lower (1.3 and 5.8 times, respectively). A similar tendency

was noted for Mn. The differences between the studied populations in terms of metal content in their leaves were less noticeable, with the exceptions of Pb and Fe (Figure 3).

**Figure 3.** Heavy metal content in the aboveground and underground organs of *N. ovata* from: (**a**) the natural forest community (P-1) and (**b**) the fly ash dump (P-2). Data is presented as mean ± SE (*n* = 5). Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

The concentration of some trace metals (Zn, Cu, Cr and Co) in the underground organs correlated with their total content in the soil (on average *r* = 0.64; Supplementary Table S1). Moreover, for copper and chromium a significant correlation was also noted with regard to the available concentration in the soil (on average *r* = 0.66, Supplementary Table S2).

The BCF for macronutrients was found in the following order at both sites: K > Ca > Mg (Table 4). Potassium was released: its average BCF in the rhizome + roots of the orchid was 42, while in the leaves was 153. The plants from FAD had increased BCF values for zinc, which were several times higher than 1 and significantly higher than in NFC. The BCF for Ni and Cr were also greater than 1, while for other studied metals were ≤1.


**Table 4.** Bioconcentration factors (BCF) and translocation factor (TF) from underground to aboveground organs of *N. ovata* from natural forest community (P-1) and fly ash dump (P-2).

#### *3.3. Morphological and Anatomical Characteristics of N. ovata*

The orchid plants growing on the fly ash substrate (P-2) had a lower shoot and inflorescence length, and number of flowers (by 30, 27, and 20%, respectively) compared to P-1, but at the same time they had a 1.4-fold larger leaf area (Table 5).

**Table 5.** Morphological characteristics of the flowering individuals of the *N. ovata* populations from the natural forest community (P-1) and the fly ash dump (P-2).


<sup>1</sup> Data is presented as mean ± SE (*<sup>n</sup>* = 20); <sup>2</sup> In the brackets are the minimum and maximum values. Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

The *N. ovata* leaves have a homogeneous mesophyll structure. The study showed that the orchids colonizing the fly ash substrate were distinguished by a thicker epidermis (by 14%) and lower mesophyll thickness (by 6%), compared to individuals from the natural habitat (Figure 4a,b).

There were no significant differences between the studied populations in terms of the number of mesophyll cells (Figure 4c), and their surface area and volume (Supplementary Table S5), whereas an increased number of chloroplasts both per cell (by 18%) and per unit cell area (by 13%), was noted for P-2 plants, compared to their P-1 counterparts (Figure 4d; Supplementary Table S5). The cell volume per chloroplast in the leaves of *N. ovata* from FAD was lower (Figure 4e) than in plants from NFC, while the reverse trend was observed for the chloroplast membrane index (Figure 4f).

**Figure 4.** The mesostructure parameters of the lower leaf of *N. ovata* from the natural forest community (P-1) and the fly ash dump (P-2): (**a**) epidermis thickness; (**b**) mesophyll thickness; (**c**) number of cells; (**d**) number of chloroplasts per cell; (**e**) cell volume per chloroplast (CVC); (**f**) chloroplast membrane index (CMI). The small solid square indicates mean values (*n* = 30); boxes present mean ± SE; the whiskers are the minimum and maximum values. Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

#### *3.4. Physiological and Biochemical Parameters of N. ovata*

As shown in Figure 5a, the leaves of *N. ovata* from the natural habitat contained 2.5 times higher Chl *a* than Chl *b*. The Chl *a* content in the P-2 plants was 1.6 times lower than in the P-1 plants. In contrast, the Chl *b* and carotenoid content in the orchids on the fly ash substrates was higher (14% and 33%, respectively). A 1.9-fold decrease in Chl *a/b* and

Chl (*a+b*)/CAR ratios was observed in plants from FAD while the (CAR+ Chl *b*)/Chl *a* ratio increased almost 2 times compared to plants from the NPC site.

**Figure 5.** The photosynthetic parameters of the leaves of *N. ovata* from the natural forest community (P-1) and the fly ash dump (P-2): (**a**) photosynthetic pigment content; (**b**) intensity of CO2 assimilation. Data is presented as mean ± SE (*n* = 12). Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

In addition, a high positive correlation between Chl *a* and available content of Cu, Ni, Fe, Mn, Mg, and Ca in soil (on average, *r* = 0.86, Supplementary Table S3) and negative correlation between Chl *a* and total Pb and Cr content in the leaves (on average, r = −0.86; Supplementary Table S4) were found for *N. ovata* plants.

The data on CO2 assimilation intensity (Figure 5b) showed that its uptake per unit area and per chloroplast per hour in P-2 plants was 1.7 times lower than in P-1 plants. At the same time, there were no significant differences between the populations in terms of the CO2 uptake per mg of chlorophyll per hour (Figure 5b).

The *N. ovata* plants on the fly ash substrate had a lower intensity of transpiration compared to the plants from the natural habitat (by 1.4 times, Figure 6a). The relative water content and water saturation deficit indexes entered the range of values of most plants and did not differ much between sites (Figure 6b). However, P-1 plants experienced a greater lack of moisture than P-2.

**Figure 6.** The water exchange parameters in the leaves of *N. ovata* from the natural forest community (P-1) and the fly ash dump (P-2): (**a**) transpiration intensity; (**b**) relative water content (RWC) and water saturation deficit (WSD). Data is presented as mean ± SE (*n* = 12). Asterisks (\*) indicate significant differences between the studied populations according to Mann–Whitney *U*-test (*p* < 0.05).

#### **4. Discussion**

The present study aims to identify the adaptive responses of the rare orchid *N. ovata* that contribute to natural colonization under the adverse conditions of a fly ash deposits. No similar investigations have been carried out in the Middle Urals. A comparative analysis of the structural and functional characteristics of *N. ovata* plants in disturbed (fly ash dump of VTTPS) and natural forest ecosystems are vital for achieving this goal.

It is well known that fly ash substrates are characterized by unfavorable physicochemical properties, which depend on the type and origin of the coal, the conditions of combustion, the type of emission control devices, and the storage and handling methods [29–31,43]. The pH values of fly ash can vary from 4.5 to 12, depending on the coal type [29]. As a rule, fly ash formed during the combustion of brown coal is alkaline [29]. The lowered pH value of the FAD substrate is obviously explained by the fact that soil formation in this area had proceeded according to the zonal type (under the conditions of a flushing water regime) [38]. According to Gajic' et al. [29], unweathered fly ash had high values of electrical conductivity (150–352 μS cm<sup>−</sup>1), which indicate a large amount of soluble salts, while EC values usually decrease (101–217 μS cm−1) in weathered fly ash. In general, plants growing during the weathering of fly ash improve the physicochemical properties of the fly ash substrate. It was found that EC of the FAD substrate was reliably lower compared to the NFC substrate. This is associated with significantly lower available concentrations of most of the studied metals in the FAD substrate, which is also confirmed by the low TDS values.

As noted, toxic concentrations of Cr, Pb, Cd, As and other metal(loid)s are often a limiting factor that reduce the rate of natural colonization of fly ash dumps [30]. The studied metal content in the soil of both sites did not exceed the maximum permissible concentrations [44]. All the studied metals, with the exception of macronutrients (Ca, Mg, and K), accumulated to the greatest degree in the roots of *N. ovata*. The increased level of trace metal accumulation in *N. ovata* roots indicates the functioning of barrier mechanisms and contributes to the implementation of an ontogenetic program [34]. Nevertheless, increased Co, Cr, Ni, and Pb content in the leaves was noted (above the normal level), which corresponds to an excessive or toxic concentration [44].

The plants growing on FAD showed the greatest difference in terms of potassium in both the aboveground and underground organs. This is due to the higher concentration of potassium in the fly ash substrate. Potassium plays a vital role in such important processes as photosynthesis, growth, assimilate transport, water exchange, etc. [45]. Thus, the high ability of *N. ovata* to absorb potassium and translocate it to leaves is one of its adaptive responses.

Fly ash substrates are known to be very poor in nitrogen content [29,30,38]. Nevertheless, its content in the orchid plants from FAD was within normal limits (at the level of average values) [45]. These results confirm the existing view that orchids can effectively assimilate nitrogen even from soils poor in this element [46]. In contrast, the total phosphorus content was higher in the leaves of *N. ovata* from FAD compared with those from NFC. Since phosphorus is involved in many biochemical, energy, and physiological processes [45], its accumulation in leaves can obviously be regarded as an adaptive response.

The *N. ovata* individuals from FAD were characterized by lower values of shoot height, inflorescence length, and the number of generative organs. These were compensated by an increase in the area of the assimilative organs.

Most orchids, especially species with thin leaves, assimilate carbon dioxide through a C3-pathway [16]. The intensity of photosynthesis depends on the activity of photosynthetic enzymes and pigment concentration [32]. On the other hand, this is largely related to the leaf blade's anatomical and morphological characteristics, which determine the optical properties and diffusion rate of CO2 to the carboxylation centers [33]. Under the stress factors, changes in the mesostructure of the photosynthetic apparatus can take place as an adaptive reaction [34,35].

The studied *N. ovata* populations revealed a lack of significant differences in leaf blade thickness. At the same time, the properties of the substrate affected the thickness of the mesophyll and epidermis: a thinner mesophyll and a thicker epidermis were characteristic of plants from FAD. This is a protective response which is probably associated with increased atmospheric dust at dump sites [29].

An increased number of chloroplasts per cell in the *N. ovata* growing in a transformed habitat can be regarded as a compensatory adaptive reaction to the lack of chlorophylls, the content of which was significantly lower in plants from the fly ash deposits. The cell volume per chloroplast is a parameter that indicates the size of the cell volume, which is provided by metabolites as well as energy substrates due to the activity of one chloroplast [34,35]. A significant decrease in this indicator in *N. ovata* from FAD, compared with NFC, is explained by an increase in the number of chloroplasts in its mesophyll cells, while the cell volume remained practically unchanged.

The chloroplast membrane index is an integral indicator of the photosynthetic apparatus [34]. Obviously, the more significant CMI observed for P-2 plants is explained by the significant increase in the number of plastids in mesophyll cells. Thus, the greater development of the leaf's internal assimilation surface in orchid plants growing on the fly ash substrate is apparently associated with lower values of tree crown density and the total projective cover of the grass-shrub layer and as a consequence, greater lighting.

It is well reported that the photosynthetic apparatus of plants ensures their vital activity in various environmental conditions [47]. The pigment complex of plants is highly sensitive to the effects of adverse factors and capable of adaptive changes. Therefore, the analysis of the content and ratio of photosynthetic pigments is of great importance in assessing the resistance of plants to various stress factors [48]. Chlorophyll *a* in *N. ovata* leaves proved to be the most sensitive to the adverse conditions in the fly ash substrate, showing significant reductions. This can be explained by a lower nitrogen content since it is one of the most important components of green pigments [49] and a high concentration of some toxic metals (perhaps, Cr and Pb). It is well known that an excess of toxic metals in plant cells can cause structural changes in chloroplasts, inhibit key enzymes in chlorophyll synthesis, and cause the destruction of pigment molecules [24,29,35].

The degree of photosynthetic apparatus activity and its resistance to unfavorable external stressors are often evaluated by the photosynthetic pigment ratio. A comparison of pigment ratios showed the decrease in the Chl *a/b* and Chl (*a+b*)/CAR ratios in plants from FAD. This fact is explained by a significantly lower Chl *a* concentration, while Chl *b* and carotenoid content were increased. The ratio of the sum of the auxiliary pigments (Chl *b* + CAR) to Chl *a*, characterizing the share of antenna forms, significantly increased in *N. ovata* plants growing in FAD. Obviously, the activation of the synthesis of auxiliary pigments is a compensatory reaction that contributes to better absorption of light for photosynthesis. However, carotenoids in chloroplasts perform not only antenna and photoprotective functions, but also an antioxidant one. Carotenoid molecules can interact with reactive oxygen species (ROS) due to double bonds [50]. Thus, an increase in the carotenoid content in plants growing on fly ash substrate is most likely a response to stress and is aimed at combating ROS.

The absence of significant changes in the assimilation index (mg CO2 mg−<sup>1</sup> chlorophyll h<sup>−</sup>1) indicates that the unfavorable conditions of the fly ash substrate did not cause significant damage to the chlorophyll molecules, since its functional ability was preserved [49].

The absorption of CO2 and the transpiration of water by plant leaves occurs through the stomata. Accordingly, the rate of CO2 assimilation and transpiration per unit area showed similar changes. The level of moisture deficiency in plants can be associated with the intensity of water evaporation. The results obtained are consistent with the data of measuring transpiration for each plot. Therefore, transpiration was more intense in orchids from NFC, than from FAD, which caused a higher water deficit. In addition, a greater moisture deficit in plants from a natural habitat can be explained by the high value of a projective vegetation cover. Consequently, there was probably competition between the plants for resources, including water.

#### **5. Conclusions**

The present study revealed those adaptive structural and functional features of *Neottia ovata* that contribute to its survival strategies in a transformed habitat. Despite the adverse edaphic conditions of the fly ash dump, the population size of this species was noticeably higher than that in natural forest community. This is due to less phytocoenotic stress on the fly ash deposits while the population of *N. ovata* in its natural habitat experiences higher level of competition. Moreover, the natural colonization of *N. ovata* on the fly ash substrate was facilitated by adaptive changes in the mesostructure parameters of the leaves, such as an increase in epidermis thickness, the number of chloroplasts in the cell, and the internal assimilating surface. The *N. ovata* plants colonizing the fly ash dump were characterized by the higher chlorophyll *b* and carotenoids content compared to plants growing in the natural forest community that evidenced the compensatory response on the decrease in chlorophyll *a*. Furthermore, *N. ovata* growing on the fly ash substrate retained a relatively favorable water balance, which also contributed to its high resistance to adverse environmental conditions.

The content of most of the studied metals in the underground parts (rhizome + roots) of *N. ovata* was considerably higher than in the leaves, which diminished the harmful effect of toxic metals on the aboveground organs, including the generative ones. Despite the lower content of most of the macro- and micronutrients in the fly ash substrate and the higher concentration of some toxic heavy metals (lead and chromium), the plants from the transformed ecosystem showed high viability.

The study of the morphophysiological features of orchids in technologically disturbed habitats is necessary for developing measures to protect the gene pool of rare plant species and to solve applied problems associated with identifying optimal conditions for naturalization and introduction into a new environment.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/horticulturae7050109/s1, Table S1: Spearman's correlation between total metal content in the soil with metals in leaves and rhizome + roots of *N. ovata* from the natural forest community and the fly ash dump; Table S2: Spearman's correlation between available metal content in the soil with metals in leaves and rhizome + roots of *N. ovata* from the natural forest community and the fly ash dump; Table S3: Spearman's correlation between total and available metal content in the soil with physiological parameters of *N. ovata* from the natural forest community and the fly ash dump; Table S4: Spearman's correlation between metal content in the leaves and rhizome + roots with physiological parameters of *N. ovata* from the natural forest community and the fly ash dump; Table S5: The mesostructure parameters of the lower leaf of *N. ovata* from the natural forest community (P-1) and the fly ash dump (P-2).

**Author Contributions:** Conceptualization, M.M., G.B. and E.F.; methodology, M.M., G.B., N.C., O.S., E.F., N.L. and M.G.; software, M.M. and O.S.; validation, M.M., G.B. and E.F.; formal analysis, M.M., G.B., E.F., N.L. and M.G.; investigation, M.M., G.B., N.C., O.S., E.F., N.L. and M.G.; resources, M.M., E.F. and O.S.; data curation, M.M., G.B. and E.F.; writing—original draft preparation, M.M., G.B. and N.C.; writing—review and editing, M.M. and G.B.; visualization, M.M. and G.B.; supervision, M.M. and G.B.; project administration, M.M., G.B. and E.F.; funding acquisition, M.M., G.B., N.C., O.S., E.F., N.L. and M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The reported study was partly funded by RFBR and the Government of Sverdlovsk region, project number 20-44-660011 and the Ministry of Science and Higher Education of the Russian Federation as part of state task of the Ural Federal University, FEUZ-2020-0057.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Acknowledgments:** The authors are very grateful to Tripti and Adarsh Kumar (UrFU, Ekaterinburg, Russia) for their constructive comments, and also Regional Center for Linguistic Support of Scientific and Publication Activity of Academic and Administrative Staff of UrFU. Two anonymous reviewers gave valuable comments for the improvement of this paper and we also gratefully acknowledged them.

**Conflicts of Interest:** The authors declare no conflict of interest.

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