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Article

Effects of Simulated Water Depth and Nitrogen Addition on Phragmites australis Root Anatomy

by
Mingyu Zhang
1,
Changwei Zhang
1,
Guijun Wang
1,
Zhenwen Xu
1,* and
Yanjing Lou
2,*
1
College of Geographical Sciences, Changchun Normal University, Changchun 130032, China
2
Jilin Provincial Joint Key Laboratory of Changbai Mountain Wetland and Ecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China
*
Authors to whom correspondence should be addressed.
Water 2026, 18(2), 243; https://doi.org/10.3390/w18020243
Submission received: 6 December 2025 / Revised: 13 January 2026 / Accepted: 15 January 2026 / Published: 16 January 2026
(This article belongs to the Section Ecohydrology)
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Abstract

Root anatomy serves as a critical indicator for understanding wetland plant adaptation strategies to environmental changes. Since water depth determines root oxygen demand while nitrogen addition regulates nutrient acquisition, the two factors exert significant and interactive effects on root anatomical structure. In this study, we established a controlled experiment employing three water depth treatments (W1: −10 cm; W2: 10 cm; W3: 30 cm), two nitrogen (N) forms (ammonium-N, nitrate-N), and four N addition levels (N0: 0 mg/L; N1: 40 mg/L; N2: 80 mg/L; N3: 160 mg/L). This design enabled us to analyze the effects of water–nitrogen interactions on the anatomical structure of reed roots to reveal wetland plants’ adaptive strategies to water-nitrogen fluctuations. The results indicate that (1) under nitrogen-free treatment, compared to the control group, the W1 treatment reduced the root aerenchyma proportion and the stele-to-root diameter ratio by 15.8% and 37.0%, respectively. In contrast, exodermis thickness increased by 32.4%, while epidermis thickness decreased by 33.7%. Under the W3 treatment, the aerenchyma proportion increased by 21.0%, the stele-to-root diameter ratio decreased by 22.2%, and exodermis thickness increased by 35.3%. (2) Compared to the nitrogen-free treatment, nitrate addition increased the root aerenchyma proportion under W1, W2, and W3 by 18.8%, 6.9%, and 18.3%. The stele-to-root diameter ratio increased by 27.9% and 12.7% under W1 and W2, but decreased by 10.8% under W3. Exodermis thickness increased by 26.3% under W2, whereas it decreased by 10.8% under W3. Epidermis thickness increased by 36.1% and 22.2% under W1 and W3, while a decrease of 12.7% occurred under W2. (3) Compared to the nitrogen-free treatment, ammonium addition increased the root aerenchyma proportion under W1, W2, and W3 by 13.6%, 13.2%, and 10.0%. The stele-to-root diameter ratio increased by 28.1% under W1 but decreased by 10.4% under W3. Conversely, exodermis thickness decreased by 20.2% under W1 while increasing by 12.6% under W3. Epidermis thickness increased by 26.3% and 20.8% under the W1 and W3 treatments. In summary, the root anatomical structure of P. australis adaptively responds to variations in water depth, nitrogen forms, and nitrogen concentrations by modulating aerenchyma proportion, the stele-to-root diameter ratio, exodermis thickness, and epidermis thickness. Future research should strengthen the study of the relationship between root anatomical traits and plant functions, to more comprehensively explore the adaptation mechanisms of wetland plants to global environmental change.

1. Introduction

Wetlands are transitional ecosystems at the land–water interface, characterized by distinct hydrological, soil, and biological properties [1]. In recent years, intensified climate change (including rising temperatures, heavy rainfall events, and atmospheric nitrogen deposition) and agricultural activities (such as irrigation and drainage of farmland, and fertilizer application) have led to more frequent flooding and increased reactive nitrogen inputs in wetlands [2]. Wetland plants often adapt to abiotic stress by modifying their morphology and structure. The root system serves as the primary organ for absorbing water and nutrients, and it is also the first organ to perceive abiotic stress [3]. The root system’s anatomical structure directly reflects its developmental stage; the presence of aerenchyma, along with epidermal thickness, exodermis thickness, and stele diameter, collectively influences a plant’s ability to resist environmental stress [4]. Therefore, investigating how root anatomical structures respond to water-nitrogen interactions will enhance our understanding of wetland plants’ adaptive strategies.
Numerous studies have demonstrated the effects of flooding and nitrogen addition levels on the anatomical structure of plant roots [5,6]. For instance, Abiko et al. [7] and Yamauchi et al. [8] found that plants can increase root aerenchyma proportion to adapt to extreme hypoxia caused by deep-water conditions. Pedersen et al. [9] further demonstrated that in rice (Oryza sativa) grown at a water depth of 80 cm, the surface area to volume ratio (SA: V) decreased by 31%, while the cortex-to-stele ratio (CSR) increased by 1.8-fold, forming the structure adapted to low oxygen conditions. Chen et al. [10] and Kirk et al. [11] demonstrated that cortical cell diameter and thickness, as well as the total area, number, and diameter of xylem vessels in plant roots, exhibit varying degrees of response under different nitrogen concentrations. Furthermore, nitrogen forms (such as NH4+ and NO3−) influence the root anatomy of wetland plants, causing them to exhibit significant differences in plastic development under anoxic conditions. For example, Gao et al. [12,13] demonstrated through lowland rice experiments that nitrate nitrogen and ammonium nitrogen exert differing effects on the formation of plant root aeration tissues under deep-water conditions. Plant preferences and responses to two nitrogen forms vary across species and environmental conditions. However, there is still a lack of systematic research on how wetland plant roots adjust their anatomical traits in response to the combined drivers of water-level fluctuations and variations in nitrogen level and form.
Phragmites australis is a dominant species in wetland ecosystems. Due to its pronounced stress tolerance, broad ecological adaptability, rapid growth, and strong reproductive capacity, it plays a crucial role in maintaining wetland biodiversity and ecosystem functioning [14]. Previous studies have examined the effects of single factors, such as water depth gradient and nitrogen concentration, on the root anatomy of P. australis. For instance, when water depth exceeds 50 cm, the root aerenchyma proportion increased from 12% to 42%, accompanied by an 18% increase in the thickness of the endodermal Casparian strip [15,16]. Eller et al. [17] further reported that under 1 m water depth, the European ecotype of P. australis exhibited a root aerenchyma proportion of 47%, compared to 35% in the Asian ecotype. In wetlands, water-level fluctuations and nitrogen inputs often co-occur. However, existing research has paid limited attention to how the root anatomy of P. australis responds to such multi-factorial interactions, which constrains our understanding of this species’ strategies for adapting to habitat changes.
Therefore, this study employed indoor simulation experiments to observe changes in the aerenchyma formation, stele diameter, cortical thickness, and epidermal thickness of P. australis roots in response to varying waterlogging depth, nitrogen form, and nitrogen concentration. The primary objectives of this study were to: (1) examine the responses of P. australis root anatomy to water-depth gradients, and (2) the responses of P. australis root anatomy to variations in water depth as well as to different nitrogen forms and concentrations. Our study will contribute to a more comprehensive understanding of the adaptation mechanisms of wetland plants to environmental changes and provide a theoretical basis for future ecological conservation and management of wetlands.

2. Materials and Methods

2.1. Study Area

In May 2023, fifty rhizomes of P. australis rhizomes were collected from the Momoge National Nature Reserve, located in the Songnen Plain of northeastern China (Figure 1). The rhizomes were immediately placed in sealed bags and stored at 4 °C. For the experiment, rhizomes of similar size were selected, surface-sterilized with a NaClO solution for 5 min, and then rinsed thoroughly with distilled water. This region experiences a temperate continental monsoon climate, with an average annual temperature of 4.4 °C and annual precipitation of 391.8 mm. Precipitation from June to August accounts for 76.6% of the annual total, resulting in pronounced seasonal fluctuations in wetland water levels. The soils were predominantly meadow and marsh types with a pH of 10.21. The vegetation is dominated by communities of Typha spp. (cattail), Scirpus spp. (bulrush), and Schoenoplectus spp. (water rush). Among these, P. australis accounts for over 60% of the plant cover and is widely distributed in shallow water and marsh areas. The Momoge Wetland, with its dense vegetation, serves as a critical habitat for wading birds and some waterfowl, providing key sites for nesting, breeding, and foraging [18].

2.2. Experimental Design

The collected P. australis rhizomes were cultivated for seedling propagation in Greenhouse No. 1 at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. The growing conditions were maintained at 25 ± 3 °C and 40–50% relative humidity. Two weeks later, seedlings approximately 25 cm in height and with uniform growth vigor were selected for subsequent experiments. The selected seedlings were transplanted individually into black plastic pots (25 cm in diameter and height) with multiple drainage holes at the bottom, totaling 150 pots. Each pot was filled with a 20 cm layer of washed river sand as the growth substrate. After transplanting, an appropriate amount of water was supplied daily to maintain the surface moisture of the sand. Following a one-week acclimatization period, plants with good growth status were chosen as the experimental material for the simulation study.
A multi-factorial interaction experiment was established in this study, with the detailed design shown in Figure 2. Based on field surveys and the hydrological characteristics of the sampling sites, three water depth treatments were established, with values relative to the soil surface: W1 (−10 cm, i.e., 10 cm below the surface), W2 (10 cm above), and W3 (30 cm above). The W1 and W2 treatments were achieved by placing pots with P. australis seedlings into white plastic containers (length × width × height: 68 cm × 47 cm × 43 cm), with five pots per container. The W3 treatment was implemented by submerging the pots in deep water buckets (diameter × height: 50 cm × 55 cm). The nitrogen (N) form and concentration gradients were designed based on field survey data, regional atmospheric N deposition levels and trends (soil total N: 2.28 mg/g; water total N: 23.58 mg/L; environmental N deposition: ~1.5 g N m−2 yr−1) [19], and established experimental designs from previous studies [20,21]. Two N forms (ammonium-N and nitrate-N) and four addition levels (N0: 0 mg/L, N1: 40 mg/L, N2: 80 mg/L, N3: 160 mg/L) were established. Combined with the three water depths, this resulted in a total of 24 treatment combinations. Each treatment was replicated five times, yielding 120 experimental units. Deionized water (containing 0.05 mg/L N-NH4, 0.21 mg/L N-NO3, and 0.05 mg/L P-PO4; average pH = 6.92) was added daily to maintain a stable water level. A half-strength nitrogen-free Hoagland nutrient solution (Table 1) was supplied to all treatments to meet essential plant nutritional requirements, with complete renewal every two weeks. To achieve target nitrogen levels, (NH4)2SO4 and Ca(NO3)2 were added at specified concentrations. Dicyandiamide (DCD) was applied at 1 mg/L as a nitrification inhibitor to maintain stable nitrogen forms.

2.3. Cryosectioning Method

The experiment lasted for 10 weeks. Upon completion, fine root samples (diameter < 3 mm) were collected from five randomly selected plants per treatment. After thorough rinsing with deionized water, a segment was excised 50 mm from the root tip and immediately fixed in a 70% FAA solution (formalin-acetic acid-alcohol) for subsequent root anatomical analysis.
A 4–6 mm long root segment was precisely excised 10 mm from the root tip using a double-edged blade and embedded with transparent adhesive for fixation. After embedding, the sample was trimmed into a regular block measuring 1.5 cm × 1.5 cm × 1 cm, ensuring that the specimen remained centered. The block was then sectioned using a cryostat microtome (model SYD-K2040, Shenyang Yude Electronic Instrument Co., Ltd., Shenyang, China), with a uniform section thickness of 25 μm. To ensure high-quality sections, at least five consecutive slices were prepared from each root sample. Following sectioning, temporary mounts were immediately prepared using optical adhesive to prevent structural distortion.

2.4. Measurements

The prepared temporary mounts were systematically examined under a Motic BA410E optical microscope (Motic Instruments Inc., Xiamen, China). Slices with intact morphology and clear tissue stratification were carefully selected for further analysis. Images were captured using the microscope’s built-in high-resolution CCD camera sequentially under 10× and 20× objective lenses. The acquired slice images were analyzed using ImageJ 1.54g to measure parameters, including total root cross-sectional area and root diameter [22]. Specifically, the aerenchyma proportion was calculated as the percentage of aerenchyma area relative to the total root cross-sectional area [23]. The stele-to-root diameter ratio was defined as the stele diameter expressed as a percentage of the root diameter [24]. All specific measurement metrics are detailed in Table 2 and Figure 3.

2.5. Statistical Analysis

Statistical analyses were performed on root anatomical data obtained from five subsamples per pot, with pot considered the experimental unit. The interactive effects of water depth, nitrogen form, and nitrogen concentration on root anatomical traits were analyzed using GLM. Specifically, a GLM with a Gaussian distribution was fitted for exodermis thickness and epidermis thickness. For proportional data (aerenchyma proportion and the stele-to-root diameter ratio), a GLM with a Beta distribution was applied. Water depth, nitrogen form, and nitrogen concentration were included as fixed effects in all models. The significance of these effects was evaluated using likelihood ratio tests. Following model estimation, Duncan’s multiple range test was applied for post hoc comparisons among nitrogen concentrations within each water depth level. All statistical analyses were performed using the R 3.6.3 [25], and figures were prepared using Origin Pro 2024.

3. Results

3.1. Effects of Water Depth Gradients, Nitrogen Forms, and Concentrations on P. australis Root Anatomy

Water depth and nitrogen concentration significantly affected all four root anatomical structures of P. australis, whereas nitrogen form only significantly influenced the aerenchyma proportion, the stele-to-root diameter ratio, and exodermis thickness (Table 3). The interactions between water depth and nitrogen form, as well as between water depth and nitrogen concentration, significantly impacted all four anatomical structures. In contrast, the interaction between nitrogen form and concentration only had a significant effect on the stele-to-root diameter ratio (Table 3).

3.2. Responses of Root Anatomical Structure to Water Depth Gradients in P. australis

Different water depth gradients had a significant effect on the development of the four measured root anatomical traits in P. australis (Table 3 and Figure 4). Compared to the control (W2), both drought (W1) and waterlogging (W3) treatments significantly altered key root anatomical traits in P. australis, including aerenchyma proportion, epidermal and exodermal thickness, and the stele-to-root diameter ratio (p < 0.05). Specifically, under W1, the aerenchyma proportion and stele-to-root diameter ratio decreased by 15.8% and 37.0%, respectively (Figure 4A,B), while exodermis thickness increased by 32.4% and epidermis thickness decreased by 33.7% (Figure 4C,D). In contrast, the W3 treatment increased aerenchyma proportion by 21.0% (Figure 4A) and exodermis thickness by 35.3% (Figure 4C), but decreased the stele-to-root diameter ratio by 22.2% (Figure 4B), with no significant change observed in epidermis thickness (Figure 4D).

3.3. Influence of Nitrate Addition on Root Anatomical Responses to Water Depth Gradients in P. australis

Both nitrogen addition and water depth treatments had significant effects on the development of the four root anatomical traits in P. australis (Table 3 and Figure 5). Compared to the nitrogen-free control, nitrate addition significantly influenced key root anatomical traits in P. australis, including aerenchyma proportion, epidermal and exodermal thickness, and the stele-to-root diameter ratio (p < 0.05). Specifically, nitrate addition increased aerenchyma proportion by 18.8%, 6.9%, and 18.3% under the W1, W2, and W3 treatments (Figure 5A). The stele-to-root diameter ratio increased by 27.9% and 12.7% under W1 and W2 but decreased by 10.8% under W3 (Figure 5B). Exodermis thickness increased by 26.3% under W2, whereas it decreased by 10.8% under W3 (Figure 5C). Epidermis thickness increased by 36.1% and 22.2% under W1 and W3, respectively, but decreased by 12.7% under W2 (Figure 5D).

3.4. Influence of Ammonium Addition on Root Anatomical Responses to Water Depth Gradients in P. australis

Variations in nitrogen form, nitrogen concentration, and water depth significantly affected the four measured root anatomical traits of P. australis (Table 3 and Figure 6). Compared to the nitrogen-free control, ammonium addition significantly influenced key root anatomical traits of P. australis, including aerenchyma proportion, epidermal and exodermal thickness, and the stele-to-root diameter ratio (p < 0.05). Specifically, ammonium addition increased the aerenchyma proportion by 13.6%, 13.2%, and 10.0% under the W1, W2, and W3 treatments (Figure 6A). The stele-to-root diameter ratio increased by 28.1% under W1 but decreased by 10.4% under W3 (Figure 6B). Exodermis thickness decreased by 20.2% under W1, while it increased by 12.6% under W3 (Figure 6C). Epidermis thickness increased by 26.3% and 20.8% under the W1 and W3 treatments (Figure 6D).

4. Discussion

4.1. Responses of P. australis Root Anatomy to Water Depth Gradients

Plant roots are key organs for perceiving environmental changes, and their anatomical structure exhibits plasticity in response to varying external conditions [26,27]. Root aerenchyma enhances plant tolerance to waterlogged conditions by forming interconnected gas spaces in the cortex, which facilitate gas exchange between aerial and belowground tissues [28]. Results showed that the aerenchyma proportion in roots decreased significantly under the W1 treatment (Figure 4A). This observation agrees with the findings of Wang et al. [29] and Yang et al. [30]. Under drought conditions, the formation of aerenchyma may reduce the efficiency of water uptake by roots; therefore, plants commonly decrease their aerenchyma proportion to acquire more water and cope with drought stress [31]. In contrast, the aerenchyma proportion in roots of P. australis increased markedly under the W3 treatment (Figure 4A), which is consistent with previous observations [32,33]. In flooded environments where oxygen availability is limited, aerenchyma serves as a pathway for oxygen transport [28,34,35,36], thereby preventing plant mortality caused by hypoxia.
The stele is the primary vascular region of the root, and its structural characteristics directly influence the efficiency of radial water and nutrient transport. Consequently, changes in stele-related parameters can effectively reflect plant anatomical responses to environmental stress [37,38]. This study found that the stele-to-root diameter ratio significantly decreased under both the W1 and W3 treatments (Figure 4B). This finding is consistent with observations by Guhr et al. [39] and Peralta Ogorek et al. [40] and likely reflects a mechanism where drought stress inhibits root apical meristem activity while promoting cortical thickening, thereby restricting the radial expansion of the vascular cylinder [41]. Under waterlogged conditions, hypoxia prompts plants to prioritize investment in aerenchyma development at the expense of root diameter growth, thereby further reducing the stele-to-root diameter ratio.
The exodermis serves as a critical barrier against radial oxygen loss (ROL). While increased exodermis thickness can reduce oxygen diffusion into the soil, excessive thickening may impede its longitudinal transport towards the root tip [15,42,43]. In this study, exodermis thickness increased significantly under both W1 and W2 treatments (Figure 4C). This aligns with findings by Wang et al. [29], who reported that P. australis roots adapt to drought stress by enhancing endodermal and exodermal development to minimize water loss [44]. Under waterlogged conditions, further strengthening of sclerenchyma cells at the exodermal edge, along with a thickened Casparian strip, effectively restricts radial oxygen diffusion, thereby reducing oxygen leakage and enhancing plant adaptation to flooding stress [45]. Furthermore, the root epidermis acts as the primary interface between the plant and soil environment, aiding in stress resistance [46]. We observed a significant 33.7% reduction in epidermis thickness under the W1 treatment (Figure 4D). A similar pattern was reported by Han et al. [47], likely because overall plant growth is constrained under drought conditions, inhibiting the division and expansion of root epidermal cells.

4.2. Effects of Nitrate Addition on the Responses of P. australis Root Anatomy to Water Depth Gradients

Nitrate addition significantly increased the aerenchyma proportion under the W1, W2, and W3 treatments (Figure 5A). The finding under drought (W1) conditions aligns with the conclusions of Gao et al. [12,13]. Ethylene is a key hormone inducing aerenchyma formation, and nitrate can enhance ethylene biosynthesis, thereby promoting aerenchyma development [35,48]. The observation under waterlogged conditions is consistent with findings in rice by Abiko et al. [7,49]. This may be attributed to the fact that in flooded environments, nitrate serves as the primary substrate for root nitric oxide (NO) synthesis, and NO is a crucial signaling molecule that induces programmed cell death in cortical cells, leading to aerenchyma formation [50].
Nitrate addition significantly increased the stele-to-root diameter ratio under the W1 and W2 treatments (Figure 5B). Nitrate can promote procambium activity, induce vascular cell proliferation, and enhance xylogenesis, leading to stele expansion [51,52,53]. Consequently, an enlarged stele improves the transport efficiency of water and mineral nutrients to the shoot and facilitates the translocation of photoassimilates to the roots to support root growth. In contrast, nitrate addition significantly decreased the stele-to-root diameter ratio under the W3 treatment (Figure 5B). This reduction likely occurred because the extreme hypoxia under W3 conditions prompted P. australis to prioritize enhancing oxygen diffusion within the roots, thereby allocating more resources to aerenchyma development. Several studies have shown that under waterlogging, root cortical thickness increases markedly, whereas inhibition of the stele is relatively less pronounced, resulting in a higher cortex-to-stele ratio [54]. Concurrently, nitrate can also promote the formation and expansion of aerenchyma [7,49], which further amplifies the increase in cortical thickness and aerenchyma proportion, ultimately causing a more pronounced decrease in the stele-to-root diameter ratio.
Nitrate addition significantly increased exodermis thickness under the W2 treatment (Figure 5C). Under well-aerated conditions, sufficient oxygen in the rhizosphere allows nitrate uptake and reduction to provide ample energy and metabolic substrates for root cells, thereby promoting exodermal cell differentiation and thickening [10,55]. In contrast, nitrate addition significantly decreased exodermis thickness under the W3 treatment (Figure 5C). This likely represents an adaptive adjustment by P. australis roots to optimize survival: under extreme hypoxia, when energy is limited and oxygen supply is deficient, plants prioritize maintaining structures critical for survival—such as aerenchyma and the vascular transport tissues of the stele—while reducing investment in comparatively less essential tissues like the exodermis, thereby optimizing resource allocation [8,9].
Nitrate addition significantly increased epidermis thickness under the W1 and W3 treatments (Figure 5D). This increase can be attributed to the ability of nitrate to promote the synthesis of cell wall components, such as cellulose, hemicellulose, and lignin, thereby thickening the epidermal cell wall [54,55]. Under drought conditions, a thicker epidermis forms a more effective physical barrier, reducing transpirational water loss. Under waterlogged conditions, nitrate further enhances epidermal wall thickening and reinforcement by stimulating nitric oxide (NO) production, which improves cellular mechanical stability and reduces flooding-induced cell death [56,57,58,59,60].

4.3. Effects of Ammonium Addition on Root Anatomical Responses of P. australis to Water Depth Gradients

Compared to the nitrogen-free treatment, ammonium addition significantly increased the aerenchyma proportion under both W1 and W3 conditions. This finding aligns with observations in rice by Chen et al. [61], who reported that ammonium can promote aerenchyma formation under drought, albeit to a lesser extent and with a delayed response compared to nitrate. This attenuated effect likely reflects the inhibitory action of ammonium on key enzymatic activities and ethylene biosynthesis [32,62], rendering NH4+ a less effective promoter of aerenchyma development. Furthermore, under drought stress, plants prioritize essential metabolism and drought-resistance mechanisms. The high energy cost associated with ammonium uptake and assimilation further limits its ability to effectively support aerenchyma formation [63]. Consequently, in arid environments, ammonium influences aerenchyma development not only as a nutrient but also through its impact on cellular energy allocation [64].
Ammonium addition significantly increased the stele-to-root diameter ratio under the W1 treatment (Figure 6B). This indicates that under drought conditions, with NH4+ as the primary nitrogen source, plants prioritize maintaining and enhancing the development of conductive tissues to improve the transport efficiency of limited water and minerals. Similarly, Pan et al. [63] demonstrated that sustained ammonium supply promotes the construction of the vascular system (stele development), thereby increasing this ratio. Furthermore, this study found that adding 40 mM ammonium significantly decreased the ratio under the W2 treatment, whereas 160 mM ammonium significantly increased it. This pattern aligns with the findings of Chen et al. [10] in Fraxinus mandshurica: low nitrogen availability leads to a significant increase in overall root diameter due to cortical thickening, reducing the stele proportion, whereas high nitrogen supply promotes vascular tissue development, increasing the stele proportion. In contrast, ammonium addition significantly decreased the ratio under the W3 treatment. This reduction can be attributed to two factors: first, NH4+ promotes the thickening of outer cortical and epidermal cell walls under hypoxia, significantly increasing the root cross-sectional diameter [65]; second, NH4+ limits stele development under waterlogged conditions, collectively leading to a significant decline in the ratio.
Ammonium addition significantly reduced exodermis thickness under the W1 treatment (Figure 6C). Under drought stress, the primary function of roots is to absorb water efficiently and rapidly. A thinner exodermis reduces the resistance to water transport across the root, thereby facilitating water uptake. Concurrently, in water-limited environments, plants often allocate limited carbon and nitrogen resources preferentially to vascular tissues to enhance stele development and water transport capacity [66], a pattern consistent with the observed increase in the stele-to-root diameter ratio in this study. In contrast, ammonium addition significantly increased exodermis thickness under the W3 treatment (Figure 6C). NH4+ promotes exodermal cell wall thickening under hypoxic conditions [65], representing a defensive adaptation. In extreme hypoxia, a thickened exodermis effectively reduces radial oxygen loss (ROL), ensuring more efficient oxygen transport from the internal aerenchyma to the root apex.
Ammonium addition significantly increased root epidermis thickness under both the W1 and W3 treatments (Figure 6D), a result consistent with the findings of Yu et al. [67]. Under drought conditions, epidermal thickening enhances the mechanical strength of the cell wall, thereby improving the root’s ability to withstand water stress. In waterlogged environments, a thickened epidermis acts as an effective barrier, serving a dual purpose: it reduces the excessive diffusion of oxygen from the aerenchyma to the external environment and prevents the entry of anaerobic microorganisms and their toxic metabolites into root tissues.

4.4. Comparison of the Effects of Water Depth Gradients and Nitrogen Addition (Form and Concentration) on Root Anatomy in P. australis

Variations in water depth gradients, nitrogen forms, and concentrations directly influence the development of plant root anatomical structures. Roots exhibit distinct adaptive mechanisms under drought and waterlogging stress. For instance, plants increase aerenchyma proportion to cope with hypoxic conditions during waterlogging, whereas the opposite trend occurs under drought [8]. Similarly, different nitrogen forms and concentrations induce plastic developmental changes in the root anatomy of wetland plants [12]. Furthermore, studies have shown that under prolonged waterlogging stress, plants prioritize water depth as the primary stressor, with nitrogen acting as a secondary factor [30]. Under drought conditions (W1), both nitrate and ammonium addition significantly increased the aerenchyma proportion, stele-to-root diameter ratio, and epidermis thickness in P. australis. In contrast, nitrate and ammonium exerted opposing effects on exodermis thickness: nitrate addition had no significant effect, while ammonium addition significantly reduced it. Under drought conditions, both nitrogen forms enhanced root water transport capacity by promoting aerenchyma and stele development, although the promoting effect of nitrate was more pronounced than that of ammonium—a finding consistent with previous studies [12,13]. Whereas nitrate addition maintained stable exodermis development, ammonium reduced exodermis thickness, potentially to optimize water absorption [68]. Under waterlogged conditions (W3), both forms increased aerenchyma proportion and epidermis thickness while decreasing the stele-to-root diameter ratio. Notably, their effects on exodermis thickness again diverged: nitrate reduced it, whereas ammonium increased it. Therefore, under waterlogging, both nitrogen forms enhance oxygen supply and reinforce the outer barrier by promoting aerenchyma and epidermis development, while reducing stele diameter to ensure safer longitudinal water transport. This divergence in their effects on exodermis thickness may stem from their differential efficacy in promoting aerenchyma development. Nitrate potently induces cortical programmed cell death and aerenchyma formation, leading to a reduction in exodermis thickness. In contrast, ammonium is less effective at stimulating aerenchyma formation; consequently, it appears to compensate by increasing exodermis thickness to minimize radial oxygen diffusion, thereby adapting to extreme hypoxia. Furthermore, genotypic variation among plants may lead to divergent expressions of root anatomical structure under stress conditions. Elucidating these genotype-specific responses offers new avenues for research aimed at clarifying how root anatomy adapts to different environmental stressors.

5. Conclusions

In this study, we investigated the anatomical responses of P. australis roots to water depth gradients, nitrogen forms, and nitrogen concentrations. The results indicate that under nitrogen-free conditions, P. australis adapts to drought by reducing aerenchyma proportion, the stele-to-root diameter ratio, and epidermis thickness, while increasing exodermis thickness. Conversely, to cope with the extreme hypoxia caused by waterlogging, it increases aerenchyma proportion and exodermis thickness but decreases the stele-to-root diameter ratio. Following nitrate addition, the development of aerenchyma and epidermis in P. australis was enhanced under both drought and waterlogged conditions. However, under waterlogging, the development of the stele and exodermis was suppressed. Following ammonium addition, aerenchyma proportion and epidermis thickness increased under both drought and waterlogged conditions. In contrast, exodermis thickness decreased under drought but increased under waterlogging. These results demonstrate that the root anatomical structure of P. australis responds plastically to variations in water depth, nitrogen forms, and nitrogen concentrations. This underscores the critical role of root anatomy in plant adaptation to environmental extremes such as waterlogging and nitrogen enrichment. Future research should prioritize linking root anatomical traits with plant functions to advance a more comprehensive understanding of how wetland plants adapt to global environmental change.

Author Contributions

Conceptualization, M.Z. and Y.L.; methodology, M.Z.; software, M.Z.; validation, M.Z., Y.L. and C.Z.; formal analysis, M.Z.; investigation, M.Z.; resources, Z.X.; data curation, M.Z.; writing—original draft preparation, M.Z.; writing—review and editing, Y.L. and G.W.; visualization, M.Z.; supervision, Y.L. and Z.X.; project administration, Y.L.; funding acquisition, Y.L. and Z.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (42171065) and the Postgraduate Innovation Project of Changchun Normal University (YJSCX2025012).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 18 00243 g001
Figure 1. Location of the rhizome sampling site in the Momoge National Nature Reserve.
Figure 1. Location of the rhizome sampling site in the Momoge National Nature Reserve.
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Figure 2. Schematic of the experimental design. (A) Nitrate treatment (NO3−-N); (B) ammonium treatment (NH4+-N), Yellow bars represent nitrate nitrogen, red bars represent ammonium nitrogen, with the number indicating concentration.
Figure 2. Schematic of the experimental design. (A) Nitrate treatment (NO3−-N); (B) ammonium treatment (NH4+-N), Yellow bars represent nitrate nitrogen, red bars represent ammonium nitrogen, with the number indicating concentration.
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Figure 3. Anatomical structure of P. australis. (magnification ×40, scale bar = 100 µm). (A) Root cross-sectional anatomy under nitrate treatment. (B) Root cross-sectional anatomy under ammonium treatment. Abbreviations: ep, epidermis; ex, exodermis; co, cortex; ae, aerenchyma; en, endodermis; st, stele. The horizontal axis represents the water depth gradient, and the vertical axis represents the nitrogen concentration.
Figure 3. Anatomical structure of P. australis. (magnification ×40, scale bar = 100 µm). (A) Root cross-sectional anatomy under nitrate treatment. (B) Root cross-sectional anatomy under ammonium treatment. Abbreviations: ep, epidermis; ex, exodermis; co, cortex; ae, aerenchyma; en, endodermis; st, stele. The horizontal axis represents the water depth gradient, and the vertical axis represents the nitrogen concentration.
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Figure 4. GLM analysis of the interactive effects of water depth gradient on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). W1: −10 cm, W2: 10 cm, and W3: 30 cm. Different lowercase letters indicate significant differences among water depth treatments (p < 0.05). Error bars represent mean ± one standard deviation (SD).
Figure 4. GLM analysis of the interactive effects of water depth gradient on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). W1: −10 cm, W2: 10 cm, and W3: 30 cm. Different lowercase letters indicate significant differences among water depth treatments (p < 0.05). Error bars represent mean ± one standard deviation (SD).
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Figure 5. GLM analysis of the interactive effects of water depth gradient and nitrate concentration on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). Nitrate concentration: NN0, 0 mg/L; NN1, 40 mg/L; NN2, 80 mg/L; NN3: 160 mg/L. Within the same water depth treatment, different lowercase letters indicate significant differences (p < 0.05) among nitrogen concentration treatments. Error bars represent mean ± one standard deviation (SD).
Figure 5. GLM analysis of the interactive effects of water depth gradient and nitrate concentration on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). Nitrate concentration: NN0, 0 mg/L; NN1, 40 mg/L; NN2, 80 mg/L; NN3: 160 mg/L. Within the same water depth treatment, different lowercase letters indicate significant differences (p < 0.05) among nitrogen concentration treatments. Error bars represent mean ± one standard deviation (SD).
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Figure 6. GLM analysis of the interactive effects of water depth gradient and ammonium concentration on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). Ammonium concentration: AN0, 0 mg/L; AN1, 40 mg/L; AN2, 80 mg/L; AN3: 160 mg/L. Within the same water depth treatment, different lowercase letters indicate significant differences (p < 0.05) among nitrogen concentration treatments. Error bars represent mean ± one standard deviation (SD).
Figure 6. GLM analysis of the interactive effects of water depth gradient and ammonium concentration on P. australis root anatomy; aerenchyma proportion (A), stele-to-root diameter ratio (B), exodermis thickness (C), and epidermis thickness (D). Ammonium concentration: AN0, 0 mg/L; AN1, 40 mg/L; AN2, 80 mg/L; AN3: 160 mg/L. Within the same water depth treatment, different lowercase letters indicate significant differences (p < 0.05) among nitrogen concentration treatments. Error bars represent mean ± one standard deviation (SD).
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Table 1. Composition of modified Hoagland’s nutrient solutions.
Table 1. Composition of modified Hoagland’s nutrient solutions.
SubstanceConcentration (μM)
KH2PO4500
KCl3000
CaCl22000
MgSO4∙7H2O1000
H3BO323.13
MnCl2∙4H2O4.57
ZnSO4∙7H2O0.382
CuSO4∙5HeO0.16
MoO30.0695
Fe-EDTA9
Table 2. Measurements of the anatomical structure of the Phragmites australis root system.
Table 2. Measurements of the anatomical structure of the Phragmites australis root system.
Tissue TypeEcological SignificanceMeasurement Index
Protective tissueMaintaining a secure interface between roots and the external environment reduces mechanical damage, pathogen invasion, and water loss.Exodermis thickness
Epidermis thickness
Vascular tissueTo achieve long-distance material transport between roots and above-ground parts, and to maintain the overall plant metabolic coordination.Stele diameter
AerenchymaRegulation of metabolism and gas exchange in roots to adapt to anoxic or resource-fluctuating environments.Aerenchyma area
Table 3. GLM results on anatomical structure of P. australis root system under different water depth gradients, nitrogen forms and nitrogen concentrations.
Table 3. GLM results on anatomical structure of P. australis root system under different water depth gradients, nitrogen forms and nitrogen concentrations.
FactorDfAerenchyma ProportionStele-to-Root Diameter RatioExodermis ThicknessEpidermis Thickness
FpFpFpFp
F127.23***1.08ns52.05***2.54ns
N311.89***17.04***8.75***24.93***
W21186.28***726.22***209.05***304.39***
F:N33.89*9.45***6.21**1.24ns
F:W241.93***33.17***203.66***16.38***
N:W618.14***44.14***8.78***14.02***
F:N:W612.79***6.82***25.26***2.12*
Notes: Nitrogen forms (F); Nitrogen treatment (N); Water depth (W). ns = non-significant. * Significant at p < 0.05. ** Significant at p < 0.01. *** Significant at p < 0.001.
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Zhang, M.; Zhang, C.; Wang, G.; Xu, Z.; Lou, Y. Effects of Simulated Water Depth and Nitrogen Addition on Phragmites australis Root Anatomy. Water 2026, 18, 243. https://doi.org/10.3390/w18020243

AMA Style

Zhang M, Zhang C, Wang G, Xu Z, Lou Y. Effects of Simulated Water Depth and Nitrogen Addition on Phragmites australis Root Anatomy. Water. 2026; 18(2):243. https://doi.org/10.3390/w18020243

Chicago/Turabian Style

Zhang, Mingyu, Changwei Zhang, Guijun Wang, Zhenwen Xu, and Yanjing Lou. 2026. "Effects of Simulated Water Depth and Nitrogen Addition on Phragmites australis Root Anatomy" Water 18, no. 2: 243. https://doi.org/10.3390/w18020243

APA Style

Zhang, M., Zhang, C., Wang, G., Xu, Z., & Lou, Y. (2026). Effects of Simulated Water Depth and Nitrogen Addition on Phragmites australis Root Anatomy. Water, 18(2), 243. https://doi.org/10.3390/w18020243

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