Influence of Water Limitation on Morphological, Physiological, and Anatomical Adaptations in Water Hyssop (Bacopa monnieri (L.) Wettst.)

1. Introduction

Water hyssop (Bacopa monnieri (L.) Wettst.) is an aquatic medicinal plant that has received considerable attention because of its diverse array of bioactive compounds with potential applications in pharmaceutical and health-related products. Previous studies have demonstrated that this plant is utilized for memory enhancement, the treatment of Alzheimer’s and Parkinson’s diseases, as well as possessing antioxidant and anti-inflammatory properties [1,2]. It possesses the ability to synthesize various chemical compounds. Among these compounds, triterpenoid saponins, particularly bacosides, are the major bioactive constituents and have been extensively investigated for their pharmacological effects, especially those associated with neurological functions [5−8]. In addition to bacosides, water hyssop contains other secondary metabolites with potential biological activities, particularly phenolic compounds and flavonoids, which have been reported to exhibit antioxidant activity and low cytotoxicity [9,10]. Moreover, polyphenol-rich extracts of water hyssop have demonstrated wound-healing activity [11]. Several phenolic compounds, including chlorogenic acid, neochlorogenic acid, and caffeic acid, have also been identified in water hyssop derived from tissue culture [12]. These findings highlight water hyssop as a promising source of bioactive compounds with potential applications in pharmaceutical and health-related products. Consequently, it serves as a vital upstream raw material for extracting bioactive compounds with significant medical applications.

However, the quantity and composition of plant secondary metabolites are determined not only by genetic factors but also strongly influenced by environmental conditions and cultivation practices. Therefore, understanding the relationship between environmental conditions, particularly water management, and plant growth and bioactive compound accumulation is essential for developing cultivation strategies that can improve the quality, consistency, and concentration of bioactive compounds in water hyssop. In medicinal plants, secondary metabolites represent key bioactive constituents with pharmacological relevance. Their biosynthesis is frequently associated with environmental stress responses [13,14]. Furthermore, the use of elicitation techniques to induce plant stress, such as drought [15], salinity, thermal, and light stress, can significantly enhance the production of secondary metabolites in medicinal plants. These controlled stress environments often result in higher yields of bioactive compounds compared to plants grown under standard field conditions or in their natural habitats [13,16−18]. Water deficit is one of the major environmental stresses affecting plant growth and physiological processes. A reduction in soil water availability can alter plant water status, stomatal regulation, gas exchange, and photosynthetic performance, with the severity and duration of water deficit playing critical roles in determining the magnitude of plant responses [19,20]. Water deficit can also disrupt photosynthetic homeostasis and promote the generation of reactive oxygen species (ROS), which have dual roles as both potentially damaging molecules involved in oxidative stress and signaling molecules that activate stress perception and adaptive responses [21,22]. The ROS-mediated responses can stimulate antioxidant defense systems and induce metabolic reprogramming, including enhanced synthesis of secondary metabolites such as phenolic compounds. These compounds can function as antioxidants and contribute to cellular protection against oxidative damage under water-deficit conditions [23,24]. Therefore, moderate water deficit may act as an eliciting stimulus that induces metabolic acclimation and promotes the accumulation of phenolic compounds. However, plant responses to water deficit are highly dependent on species, stress intensity, and stress duration. When water deficit becomes too severe or prolonged, the capacity for acclimation may be exceeded, resulting in impaired photosynthetic activity, increased oxidative damage, and reductions in biomass production [20,24].

The Water hyssop proliferates quickly in wetlands [26]. It naturally associated with moist and water-abundant habitats, the capacity to acclimate to declining soil water availability and the relationship between water-deficit intensity and phenolic accumulation remain insufficiently understood. In particular, the soil-moisture level and duration of water restriction required to achieve a balance between physiological acclimation and enhanced accumulation of bioactive compounds have not been clearly established. Identifying an optimal stress level is more informative than simply assessing growth inhibition or physiological damage. Controlled water limitation could potentially be used as a cultivation tool to stimulate secondary metabolite production without compromising plant growth or photosynthetic performance. This approach could provide a basis for precision water management aimed at enhancing the accumulation of valuable bioactive compounds in water hyssop. Therefore, this study aimed to characterize the physiological and adaptive responses of water hyssop to different levels and durations of soil water restriction by evaluating changes in morphology, water status, photosynthetic performance, oxidative stress, total phenolic content (TPC), and anatomy. The study particularly emphasized identifying the relationships among soil water availability, physiological acclimation, and phenolic accumulation. These findings are expected to define a suitable water-deficit window that stimulates phenolic accumulation without causing severe physiological damage, and to provide a scientific foundation for developing water-management strategies to produce water hyssop with enhanced phenolic content and consistent quality.

2. Results and Discussion

2.1. Effect of Soil Moisture Depletion on Morphological and Phisiological Changes

Water hyssop (Bacopa monnieri) is an aquatic medicinal plant commonly found along riverbanks and moist habitats, thriving under high soil moisture conditions. Consequently, water deficit imposes severe environmental stress on this species, triggering pronounced morphological and physiological responses. The study found that reducing soil moisture by withholding water significantly affected the morphological characteristics of water hyssop (Figure 1). Visual monitoring of the plantlets and soil surface conditions over 12 days drought induction period revealed distinct physiological responses as the water deficit intensified. At the onset of the experiment, the plantlets exhibited optimal health and vigor. The leaves were vibrant green, and the stems remained turgid, upright, and succulent. The soil surface was saturated, characterized by a dark color and a complete absence of surface cracking. For early stress phase (day 1–6), as the duration of water deprivation increased, the overall appearance of the plant remained relatively similar to the initial state. However, subtle changes began to emerge, such as a slight leaning or loss of verticality in some stems. Simultaneously, the soil began to lose its moisture content. This was indicated by a noticeable lightening of the soil color and a drying of the surface layer. Despite these changes, no significant or clearly visible soil cracking had developed by the end of this period.

For intermediate stress phase (day7–9), symptoms of wilting became increasingly pronounced. Some leaves began to turn yellow (chlorosis), reflecting a clear physiological response to water deficit, although the stems remained green. During this period, the soil became extremely dry, with a significantly lighter color. Initial surface cracking began to appear in certain areas of the soil.

By the end of the experiment (days 10–12), Water Hyssop exhibited severe wilting. The leaves transitioned from yellow to brown, becoming brittle and desiccated after 12 days. These symptoms indicated a progression toward permanent wilting point and potential plant death. The soil conditions deteriorated into a hard, pale, and arid state, with deep and distinct cracks visible across the entire surface of the pot.

For quantitative analysis of soil moisture and relative water content (RWC) showed that the 12-day water deprivation experiment demonstrated a continuous decline in both soil moisture and the plant’s RWC. Statistical analysis revealed distinct physiological phases in response to the progressing drought. During the initial phase (days 0–1), soil moisture remained high at 82.89% and 85.36%, respectively. Day 2, a statistically significant drop occurred, with moisture levels falling to 66.12%. Day 7, moisture content plummeted to 21.86%. Days 7–12, following the sharp decline on day 7, soil moisture continued to decrease but showed no further statistical differences during this late stage, reaching a minimum of 6.54% on day 12. (Figure 2a). In contrast to the rapid decline in soil moisture, the RWC of water hyssop remained stable during the first week (Figure 2b). Days 0–7, RWC stayed consistently high (ranging from 95.09% to 89.88%) with no significant variations, despite the significant reduction in soil moisture starting on Day 2. Days 8–12, Once soil moisture fell below the 21.86% threshold, the RWC began to drop drastically: 78.50%, 57.73%, 35.24%, 27.25%, and 11.15%, respectively. (Figure 2a and Figure 2b). The decline in soil moisture also resulted in reductions in photosynthetic performance, as indicated by decreases in the maximum quantum efficiency of photosystem II (Fv/Fm) and electron transport rate (ETR). For the impact of drought stress on photosynthetic efficiency showed that continuous water deprivation significantly affected the maximum quantum yield of photosystem II (Fv/Fm) and the electron transport rate (ETR) of water hyssop. From day 1 to day 9 of the drought treatment, Fv/Fm values remained statistically stable, ranging between 0.6626 and 0.7438. This suggests that the structural integrity of Photosystem II (PSII) was maintained despite the decline in soil moisture (starting from day 2) and the reduction in RWC (starting from day 8). However, a critical collapse occurred on day 10, where Fv/Fm plummeted to 0.2475, a statistically significant drop compared to previous days. While soil moisture levels between day 9 and day 10 were not statistically different, the RWC dropped sharply from 57.73% to 35.24% during this interval. This severe internal water loss resulted in catastrophic damage to the PSII reaction centers. By days 11 and 12, Fv/Fm became undetectable due to the total failure of the photosynthetic system (Figure 2c). The ETR data followed a similar trend to Fv/Fm. During the initial phase (days 0–9), the ETR values fluctuated slightly but remained statistically insignificant, with an average of 11.65 µmol m^-2s^-1. By Day 10, the ETR decreased significantly to 0.45 µmol m^-2s^-1, indicating a near-complete inhibition of electron transport. The ETR could no longer be measured on Days 11 and 12, signifying a total cessation of the light-dependent reactions (Figure 2d). Similar responses have been reported in plants exposed to drought stress, although the magnitude and direction of changes in chlorophyll fluorescence parameters depend on plant species, stress intensity, and stress duration [26]. Some plants may initially maintain or even increase photosynthetic activity during the early stages of soil drying before experiencing a progressive decline as drought severity increases. Under severe drought, pronounced reductions in chlorophyll fluorescence parameters and ETR indicate impairment of photosynthetic electron transport and PSII function [27]. In the present study, F_v/F_m values of water hyssop under well-watered conditions were already below the commonly reported range of 75–85% for non-stressed plants [28]. Furthermore, none of the soil moisture treatments resulted in F_v/F_m values exceeding 0.75 (Figure 2c). Therefore, the commonly cited F_v/F_m threshold should not be directly applied as an absolute criterion for defining drought stress in water hyssop. Instead, changes relative to the species-specific baseline under well-watered conditions provide a more appropriate indicator of PSII responses to drought stress. A pronounced decline in both F_v/F_m and ETR occurred when soil moisture decreased to 9.10%, whereas these parameters remained relatively stable at higher soil moisture levels (Figure 2a, Figure 2c and Figure 2d). This finding suggests that 9.10% soil moisture may represent a critical threshold at which photosynthetic performance becomes substantially impaired in water hyssop. In contrast, plant water status, as indicated by RWC, began to decline at a considerably higher soil moisture level of 21.86% and continued to decrease progressively with further soil drying.

Continuous water deprivation induced varying levels of physiological stress in water hyssop, as indicated by the accumulation of Hydrogen peroxide (H₂O₂) and Malondialdehyde (MDA) (Figure 2e and Figure 2f). For H₂O₂ accumulation, during the initial phase of the drought (days 0–9), H₂O₂ levels showed no statistically significant differences, with an average accumulation of 86.51 µM. Day 10, a rising trend was observed as H₂O₂ levels increased to 130.90 µM. Days 11 and 12, the levels of H₂O₂ surged dramatically to 195.80 and 209.76 µM, respectively. These values represent a statistically significant increase compared to the previous days, signaling severe oxidative stress (Figure 2e). The accumulation of MDA followed a similar pattern to that of H₂O₂. Days 0–10, there were no significant differences in MDA levels, which averaged 15.96 nmol g^-1 fresh weight. Days 11 and 12, MDA concentrations reached peak levels of 45.37 and 39.98 nmol g^-1 fresh weight, respectively. These levels were significantly higher than those recorded during the first nine days of the experiment (Figure 2f). The results indicate that prolonged water deprivation triggers a massive accumulation of H₂O₂ in water hyssop tissues. This rise in reactive oxygen species (ROS) directly correlates with the significant increase in MDA levels on days 11 and 12. The elevated MDA concentrations reflect severe lipid peroxidation, which indicates that the cell membrane integrity has been compromised. This data confirms that the severity of cellular damage intensifies as the period of water deprivation is extended, ultimately leading to the physiological failure of the plant. The H₂O₂ is an important ROS that can function as both a signaling molecule and, when excessively accumulated, a source of oxidative damage to cellular components. MDA is a major product of lipid peroxidation and is widely used as an indicator of oxidative membrane damage under abiotic stress [29].

For total phenolic content (TPC), during the initial period of water deprivation (days 0–8), the TPC remained statistically stable, with an average concentration of 92.20 µg mL^-1. Day 9, a rising trend emerged as TPC increased to 125.59 µg mL^-1. Day 11, the TPC reached its peak concentration of 195.69 µg mL^-1 when soil moisture dropped to 9.06%. Day 12, as soil moisture reached its minimum of 6.54%, TPC levels began to decline slightly. However, these levels remained elevated compared to the early stages of water deprivation (Figure 2g). The results demonstrate that reducing soil moisture effectively stimulates the synthesis of phenolic compounds. This response is a key component of the plant’s antioxidant defense mechanism, designed to mitigate cellular damage during drought stress. The peak TPC value at 9% soil moisture suggests that moderate to severe stress acts as a catalyst for metabolite production. However, the subsequent decline in TPC when moisture fell to 7% indicates that extreme stress may impair the plant’s metabolic capacity or biosynthetic pathways.

Drought stress serves as a powerful elicitor for the biosynthesis of secondary metabolites in water hyssop. Given that this species naturally thrives in riparian and wetland habitats, water deprivation triggers significant morphological and physiological changes. Visual symptoms of drought stress include wilting and a distinct color transition from green to yellow and brown. At 9.06% soil moisture, the leaves exhibit severe wilting and turn brown; however, the stems remain green. At this specific stage, the plant reaches its peak capacity for phenolic compound synthesis. At 7% soil moisture, the entire plant becomes desiccated and brown, leading to a marked decline in phenolic production. This suggests that while moderate stress acts as a catalyst, extreme desiccation impairs metabolic pathways. Phenolic compounds are important non-enzymatic antioxidants that contribute to the scavenging of reactive oxygen species (ROS) and the maintenance of cellular redox homeostasis under drought stress [30]. Increased phenolic accumulation has also been associated with enhanced stress tolerance in several plant species [31]. However, this response is highly dependent on the type of phenolic compound, plant tissue, stress duration, stress intensity, and plant species. Therefore, an increase in total phenolic content (TPC) should not be considered a universal indicator of drought tolerance. Under prolonged or severe drought stress, phenolic accumulation may decline as metabolic activity and cellular integrity become increasingly impaired [32]. The present results are consistent with this concept, as TPC increased as drought stress intensified but declined at the most severe level of water deficit.

The divergence between the responses of RWC and photosynthetic parameters suggests that water hyssop can tolerate a considerable decline in plant water status before pronounced impairment of PSII photochemistry and electron transport occurs. However, once soil moisture declined to 9.10%, both F_v/F_m and ETR decreased markedly, indicating that the photosynthetic apparatus had reached a critical level of functional impairment. Importantly, TPC remained high at approximately 9.05% soil moisture, suggesting that the plant continued to allocate metabolic resources toward antioxidant defense despite the substantial decline in photosynthetic performance. This response may indicate a shift in physiological priorities from growth and energy acquisition toward cellular protection under severe water deficit.

At the most severe drought conditions, corresponding to 6.54% soil moisture, the accumulation of phenolic compounds also declined, coinciding with severe morphological deterioration and extensive tissue dehydration. This pattern suggests that the protective metabolic response observed under moderate-to-severe drought stress could not be sustained under extreme water deficit. Collectively, these findings indicate that water hyssop exhibits a coordinated drought response involving progressive water loss, impairment of photosynthetic performance, oxidative stress, and enhanced phenolic accumulation. However, when drought severity exceeds a critical threshold, both photosynthetic function and secondary-metabolite accumulation become compromised, ultimately leading to severe morphological damage and loss of physiological function. An important finding is that while the photosynthetic system begins to fail at 9.05–9.10% soil moisture, the synthesis of phenolic compounds peaks at this exact stage. This indicates a strategic metabolic shift: the plant diverts its remaining energy away from primary growth (photosynthesis) and toward defense mechanisms (secondary metabolite synthesis) to mitigate cellular damage.

In the present study, total phenolic content (TPC) positively correlated with H₂O₂ (r=0.89) and MDA (r=0.86), indicating that increasing oxidative pressure was associated with enhanced accumulation of phenolic compounds (Figure 3). Drought stress activates the phenylpropanoid pathway to generate antioxidant metabolites, explaining the TPC increase under moderate-to-severe drought as an adaptive defense to mitigate cellular damage. However, the TPC drop under extreme drought suggests that severe metabolic damage eventually overwhelms this protective response.

Among the physiological parameters evaluated, Relative Water Content (RWC) was found to be the most sensitive indicator of decreasing soil moisture in water hyssop. Once the internal water content reached a critical threshold, it triggered a significant decline in photosynthetic efficiency, which directly correlated with the accumulation of oxidative stress markers. This relationship was statistically confirmed by the strong negative correlation between Fv/Fm and both H₂O₂ (r = -0.94) and MDA (r = -0.84). Similarly, the Electron Transport Rate (ETR) exhibited a high negative correlation with H₂O₂ (r = -0.82), while showing a moderate negative correlation with MDA levels (r = -0.61) (Figure 3).

2.2. Effect of Prolonged Drought Stress

2.2.1. Morphologcal Response

Water hyssop plant began to exhibit clear physiological responses when soil moisture declined to approximately 21.86%, while the highest phenolic accumulation (195.69 µg mL^-1) occurred at 9.05% soil moisture, indicating a critical water-deficit level that strongly stimulated phenolic biosynthesis. Based on these responses, plants were maintained at 20-30% soil moisture for 0-4 weeks and subsequently exposed to 8-10 soil moisture to assess drought acclimation and induce phenolic accumulation. After four weeks, plants maintained at 20-30% soil moisture for 0-2 weeks remained largely green and vigorous, whereas those exposed for 3 weeks exhibited lighter green, soft and wilted leaves with fewer leaves, and those exposed for 4 weeks showed extensive leaf browning, although newly formed branches remained green; stem appearance was relatively unchanged among treatments (Figure 4a). Following exposure to 8-10% soil moisture, non-acclimated plants (0 weeks) exhibited the most severe wilting and desiccation, whereas plants previously maintained at 20-30% soil moisture for 1-4 weeks retained more green tissues, although some leaves became dry and brown, suggesting that prior exposure to moderate water deficit enhanced subsequent drought tolerance. Prolonged exposure to 20-30% soil moisture also significantly reduced branch and leaf numbers, particularly after 2-4 weeks, whereas main-shoot length and leaf size remained statistically unchanged (Figure 4). These results indicate that prolonged water limitation primarily altered shoot architecture rather than main-shoot elongation. Reduced leaf production under water deficit is consistent with the restriction of cell expansion and division caused by reduced water potential and turgor, while reduced leaf number can also function as a drought-avoidance strategy by limiting transpirational surface area and water loss [33,34]. Similarly, the decline in branching may reflect drought-induced changes in hormonal signaling and resource allocation, as shoot branching is regulated by coordinated interactions among auxin and cytokinin [35,36], while increased abscisic acid (ABA) signaling under water deficit promotes stomatal closure and restricts shoot growth as part of the plant drought-response network [33,34]. Thus, the maintenance of main-shoot length and leaf size despite reduced branch and leaf production suggests that water hyssop responds to prolonged low soil moisture by reallocating resources toward water conservation, reducing the formation of new transpiring organs while maintaining existing shoot structure.

2.2.2. Physiological Responses

Water hyssop plant exhibited a clear time-dependent physiological response to prolonged low soil moisture (20–30%). Relative water content (RWC) and leaf greenness remained relatively stable under low soil moisture for 1–2 weeks but declined markedly under low soil moisture for 3–4 weeks, indicating that water hyssop initially maintained leaf water status and photosynthetic capacity but experienced increasing water limitation under prolonged stress. Consistently, photosynthetic responses were dynamic rather than progressively declining: F_v/F_m and electron transport rate (ETR) decreased under water stress for 2–3 weeks, whereas F_v/F_m recovered to a level comparable with well-watered plants under water stress for 4 weeks. This transient reduction followed by recovery suggests that the photosynthetic apparatus was temporarily inhibited but subsequently acclimated to prolonged water limitation, thereby maintaining PSII function rather than undergoing irreversible damage [37,38]. Prolonged stress also increased H₂O₂ accumulation after 3–4 weeks, whereas malondialdehyde (MDA) remained unchanged (Figure 5a-f), suggesting that elevated reactive oxygen species (ROS) were effectively controlled and did not result in substantial membrane lipid peroxidation. Moreover, H₂O₂ may have functioned not only as an oxidative stress indicator but also as a signaling molecule involved in stress perception, gene regulation, hormonal signaling, and activation of antioxidant defenses [39,40]. Collectively, these findings indicate that water hyssop undergoes a coordinated, time-dependent acclimation response to prolonged water limitation, characterized by an initial maintenance of water status and photosynthetic function, transient photochemical inhibition during intermediate stress, and subsequent photosynthetic acclimation accompanied by effective control of oxidative damage. Such physiological plasticity may contribute to the ability of water hyssop to maintain cellular and photosynthetic function under prolonged soil moisture deficit.

2.2.3. Biomass Production and Phenolic Accumulation

Maintaining soil moisture at 20–30% for 1–3 weeks induced no significant alterations in the fresh or dry biomass of water hyssop relative to the well-watered control. Conversely, extending water limitation to 4 weeks significantly diminished fresh weight, while subsequent exposure to severe water deficit (8–10% soil moisture) markedly reduced dry biomass accumulation (Figure 6a). These results indicate that water hyssop can maintain biomass production under moderate water deficit for a limited period, but prolonged stress eventually constrains growth, consistent with the well-maintained relative water content (RWC) during the first 1–2 weeks and its significant decline after 3–4 weeks of water restriction. Interestingly, plants pre-exposed to water restriction for 1 week exhibited higher fresh weight than both the control and other water-restriction treatments after soil moisture was reduced to 8–10%, while dry weight remained comparable to the control in plants restricted for 1–3 weeks. Together with the RWC response, this pattern may indicate an acclimation response or stress priming, whereby moderate prior stress enhances the plant’s capacity to cope with subsequent severe drought through adjustments in stress signaling, redox homeostasis, and metabolism [41−44]. In contrast, prolonged water restriction for 4 weeks resulted in reduced dry biomass, suggesting that the physiological costs of sustained stress eventually exceeded the plant’s adaptive capacity. In terms of secondary metabolism, water restriction for 2–3 weeks significantly increased total phenolic content (TPC), whereas 1 and 4 weeks of restriction did not differ from the control, indicating that phenolic accumulation was dependent on both stress intensity and duration rather than increasing linearly with drought duration. Moderate water deficit may stimulate secondary metabolism and antioxidant defense through ROS-mediated signaling and the activation of phenylpropanoid metabolism [24,45], whereas the absence of further TPC accumulation after 4 weeks may reflect metabolic acclimation or a shift in resource allocation toward cellular homeostasis and survival [23]. Following severe water deficit at 8–10% soil moisture, TPC did not differ among plants subjected to different durations of prior water restriction, with a maximum value of 192.60 µg mL⁻¹, comparable to 195.69 µg mL⁻¹ observed in the previous experiment (Figure 6b). This result suggests that, under the present experimental conditions, phenolic accumulation approached a plateau and that increasing drought severity did not further enhance TPC. Excessive or prolonged water deficit may instead impose limitations on carbon availability, energy status, and metabolic activity required for secondary metabolite biosynthesis [46]. Overall, these findings suggest that moderate water restriction at 20–30% soil moisture for 2–3 weeks represents a potentially optimal stress window for enhancing phenolic accumulation in water hyssop while maintaining biomass production, whereas more severe or prolonged stress provides limited additional benefit and may compromise plant productivity.

2.2.4. Anatomical Acclimation

Although water hyssop sustained growth under moderate water limitation (20–30% soil moisture) for up to four weeks, prolonged restriction triggered pronounced physiological and anatomical adjustments in both leaves and stems. (Figure 7). Under water limitation, leaf epidermal and sub-epidermal cells exhibited reduced cell area. Additionally, structural modifications were observed in the vascular tissues and mesophyll layer, characterized by cellular flattening and a loss of regular conformation relative to the control group (Figure 7a-b). An increase in mesophyll intercellular spaces and a marked reduction in chloroplast abundance were also observed, consistent with the decline in relative water content (RWC) and leaf greenness, together with the tendency toward lower Fv/Fm and ETR, indicating impairment of plant water status and photosynthetic performance under prolonged water limitation (Figure 7-b). Reduced cellular turgor under limited water availability may restrict cell expansion and contribute to the observed anatomical modifications, whereas changes in vascular bundle and xylem structure may represent adjustments of the water-conducting system to maintain leaf water balance [47,48]. The increased mesophyll intercellular spaces may further reflect structural acclimation to water limitation and potentially influence CO₂ diffusion and photosynthetic performance [49]. Following 4 weeks of water restriction, leaf anatomical observations showed reduced chloroplast abundance (Figure 7b). This decline correlated with decreased photosynthetic performance and increased H₂O₂ concentration. Nevertheless, MDA content remained statistically unchanged from controls, suggesting that oxidative stress occurred without inducing severe membrane lipid peroxidation [49,50].

In stems, water limitation reduced intercellular spaces in the cortex, altered the shape of cortical cells adjacent to vascular bundles, enhanced starch grain abundance around vascular bundles, and markedly decreased xylem vessel size. (Figure 7c-h). The reduced intercellular spaces and altered cortical cell morphology may be associated with restricted cell expansion under reduced water availability, whereas increased starch accumulation may reflect changes in carbohydrate storage or allocation as growth becomes constrained, rather than a direct enhancement of phenolic biosynthesis, as TPC at 4 weeks did not differ from the well-watered control. Furthermore, the reduction in xylem vessel size observed in both leaves and stems may represent an anatomical adjustment to declining water availability, potentially contributing to hydraulic safety at the expense of hydraulic transport efficiency [51−53]. Collectively, the coordinated changes in leaf mesophyll structure, vascular tissues, chloroplast abundance, and starch storage demonstrate that water hyssop employs multiple levels of anatomical and physiological acclimation to sustained drought. These adjustments help preserve tissue hydration and physiological function despite severe water limitation.

3. Materials and Methods

3.1. Plant Material and Experimental Set-Up

Healthy mother plants of Bacopa monnieri were collected from their natural habitat at Faculty of Fisheries, Kasetsart University, Thailand. The voucher specimen of B. monnieri (voucher no. 084795) was deposited in the Bangkok herbarium, Department of Agriculture, Thailand. Shoot cuttings of water hyssop (B. monnieri) approximately 10 cm in length were propagated in peat moss for 2 weeks to facilitate root development. The rooted cuttings were subsequently transplanted into pots containing well-moistened soil. To ensure high soil moisture, pots were placed in trays inside the greenhouse for two weeks. Greenhouse conditions were set to 30±2 °C with 10 h per day of photosynthetically active radiation (PAR) at 37 µmol m^–2s^–1.

3.2. Drought Stress Treatments and Experimental Design

Two drought-stress experiments were conducted. The first experiment was performed to determine the soil moisture threshold that induced water stress and promoted the accumulation of bioactive compounds, particularly phenolic compounds. The second experiment was conducted to investigate plant adaptation to prolonged drought stress.

Experiment 1, drought stress was initiated by removing all pots from the trays, thereby allowing soil moisture to gradually decrease under greenhouse conditions. Plant samples were randomly collected at daily intervals following the initiation of water-deficit conditions until visible wilting symptoms developed. The soil moisture level associated with the onset of visible wilting was subsequently used to define the drought-stress threshold for the subsequent experiment.

Experiment 2, soil moisture was maintained at 20–30%, as determined from the first experiment, for 0, 1, 2, 3, or 4 weeks. Following each drought-treatment period, soil moisture was adjusted to 8–10%, corresponding to the soil moisture level associated with the highest total phenolic compound content identified in the first experiment, and maintained until harvest (Figure 8).

3.3. Morphological Study and Biomass Measurements

The morphological characteristics of water hyssop were evaluated using random samples from the hydroponic tray system post-culture. We examined structural features under a stereomicroscope with a camera (Olympus SZ 40) and measured shoot length, number of branches, leaf number, and leaf area. Daily tracking, supported by representative images. Fresh weight and dry weight were determined at 4 weeks after the initiation of drought stress and when soil moisture reached 8–10%.

3.4. Assessment of Physiological Parameters

Physiological parameters were measured daily during the first experiment from the initiation of drought stress until the plants exhibited visible wilting symptoms. In the second experiment, the same parameters were measured at 0, 1, 2, 3, and 4 weeks after the initiation of drought stress. The measured parameters included soil moisture content, relative water content (RWC), leaf greenness, photosynthetic efficiency, hydrogen peroxide (H₂O₂) content, and malondialdehyde (MDA) content. Leaf greenness was quantified using a portable chlorophyll meter (SPAD-502; Minolta Corp., Kyoto, Japan) on the first to third fully expanded leaves of each plant. Photosynthetic performance and RWC were assessed using the same leaves. Photosynthetic performance was measured using a pulse-amplitude-modulated fluorometer (PAM-2500; Walz, Effeltrich, Germany). The maximum quantum efficiency of photosystem II (Fv/Fm) and electron transport rate (ETR) were recorded. The RWC was determined according to the following equation (1) [54]:

Leaf RWC (%) = [(FW-DW) / (TW-DW)] x 100

(1)

where FW is the fresh weight, TW is the turgid weight, and DW is the dry weight.

Hydrogen peroxide (H₂O₂) content was determined following a modified method of [55] Briefly, 0.1 g fresh tissue was homogenized in 1.5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 12,000 × g for 15 min at 4 °C. An aliquot of 0.5 mL of the supernatant was mixed with 0.5 mL of potassium phosphate buffer (pH 7.0) and 1 mL of potassium iodide solution. The reaction mixture was incubated at room temperature for 20 min. Absorbance was measured at 390 nm using a microplate reader. H₂O₂ concentration was calculated based on a standard calibration curve.

Malondialdehyde (MDA) Assay, lipid peroxidation was estimated by determining MDA content [55]. Sample (0.1 g) was ground in liquid nitrogen and homogenized in 2 mL of 0.1% (w/v) TCA. The homogenate was centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was mixed with 1 mL of 0.5% (w/v) thiobarbituric acid (TBA) solution and incubated at 98 °C for 30 min. The reaction was terminated by cooling to room temperature. Absorbance was measured at 532 and 600 nm MDA concentration was calculated using the following equation (2):

MDA (µmol g⁻¹ FW) = [(A₅₃₂ − A₆₀₀) / 155] × (V_TCA / V_sample) × (V_sample + V_TBA) / W

(2)

where: V_TCA is the volume of TCA used for extraction (mL), V_sample is the volume of extract used for reaction (mL), V_TBA is the volume of TBA solution added (mL), and W is the fresh weight of plant sample (g)

3.5. Quantification of Total Phenolic Content

Total phenolic content (TPC) was determined daily during the first experiment. In the second experiment, TPC was determined after 4 weeks of drought treatment and again when soil moisture reached 8–10%. The TPC was evaluated using a modified Folin–Ciocalteu method based on [56]. Briefly, 0.1 g of dried plant material was extracted with 10 mL of 95% ethanol and incubated for 12 h. Subsequently, 0.2 mL of the extract was mixed with 1 mL of 10% Folin–Ciocalteu reagent and thoroughly vortexed. The mixture was incubated at room temperature for 8 min, followed by the addition of 0.8 mL of 20% sodium carbonate (Na₂CO₃). The reaction mixture was then incubated in the dark at room temperature for 90 min. Absorbance was measured at 765 nm using a spectrophotometer. TPC was quantified against a gallic acid calibration curve and expressed as gallic acid equivalents (GAE).

3.6. Anatomical Analysis

Anatomical characteristics were evaluated after 4 weeks of drought stress with soil moisture maintained at 20–30%. Samples of stems and leaves were collected for anatomical analysis and prepared as permanent sections adapting from [57] The samples were initially fixed in FAA solution for at least 24 h. The specimens were then trimmed in distilled water and post-fixed in 2.5% glutaraldehyde prepared in 0.1 M sodium phosphate buffer (pH 7.2) for 24 h, followed by three washes with 0.1 M sodium phosphate buffer (pH 7.2).

The samples were subsequently post-fixed in 2% osmium tetroxide for 2 h and washed three times with distilled water. Dehydration was performed using a graded acetone series (20%, 40%, 60%, and 80%), followed by three changes of 100% acetone, with each step performed for 15 min. The dehydrated samples were infiltrated with Spurr’s resin by gradually increasing the acetone-to-resin ratio to 75:25, 50:50, 25:75, and 0:100 (v/v), with each step performed for 30 min. The samples were then embedded in molds and polymerized at 80 °C.

Semi-thin sections (1 μm thick) were prepared using an ultramicrotome (EM UC7, Leica, Vienna, Austria). For staining, the sections were transferred onto glass slides containing a drop of distilled water and placed on a hot plate until the water evaporated. The sections were stained with 1% toluidine blue for 5 min and subsequently rinsed with distilled water. The slides were then placed on a hot plate until the sections became clear and were mounted as permanent preparations. Anatomical characteristics were examined and photographed using a compound light microscope (Axioscope 5, Carl Zeiss, Jena, Germany).

3.7. Statistical Analysis

Comparisons of the data between the treatments were carried out based on Tukey’s multiple comparisons test, with significance tested at p≤0.05 using SPSS statistical software (SPSS Inc., Chicago, IL, USA). Results were expressed as mean ± standard deviation (SD) values.

4. Conclusions

In this research, it can be concluded that progressive water deficit stimulates an integrated response across morphological, physiological, and anatomical adaptations in water hyssop.

Morphologically, reduced branching and new leaf production may have limited the development of additional transpiring surface area, while main shoot length and leaf size were maintained, suggesting a balance between restricting water loss and sustaining shoot growth.

Physiological responses became evident at 21–22% soil moisture, whereas 8–9% soil moisture acts as a critical threshold at causing severe photosynthetic performance by increased oxidative stress and cellular damage. In response, water hyssop enhanced phenolic accumulation which may contribute to protection against oxidative stress. Short-term water limitation for 2 weeks stimulated TPC accumulation, whereas TPC declined to a level comparable with the well-watered control after 4 weeks of prolonged stress. Prolonged water limitation was also associated with changes in plant water status, leaf greenness, and photosynthetic performance, accompanied by anatomical modifications, including increased intercellular spaces in the leaf mesophyll, reduced intercellular spaces in the stem cortex, altered vascular bundle morphology, reduced chloroplast abundance, smaller stem xylem vessels, and increased starch accumulation in the cortex.

Collectively, these responses indicate that water hyssop exhibits coordinated morphological, physiological, and anatomical adjustments to maintain water balance. The results provide a basis for optimizing irrigation management and controlled stress cultivation to produce water hyssop biomass enriched in bioactive compounds without compromising photosynthetic performance and growth.