Electrophysiological Insights into the Adaptability of Bletilla striata to Bicarbonate Stress in Karst Habitats

Abstract

Bletilla striata, a perennial orchid of both medicinal and ecological value, exhibits remarkable adaptability to bicarbonate-rich karst environments. To elucidate its physiological and electrophysiological responses to bicarbonate stress, seedlings were cultivated for 45 days under NaHCO₃ concentrations of 0, 5, 10, and 15 mM (n = 4), with the nutrient solution renewed daily. At 5 mM, biomass, chlorophyll content, electrophysiological traits, nutrient transport, metabolic indices, and conductance–resistance parameters did not differ significantly from controls, while intracellular water-use efficiency exhibited only a minor, non-significant increase—indicating stable physiological performance under low bicarbonate conditions. By contrast, higher concentrations (≥10 mM), particularly 15 mM, markedly reduced intracellular water-holding capacity (−35.90%), nutrient translocation capacity (−22.26%), and metabolic activity (−50.00%), alongside electrophysiological signatures of diminished capacitance (−48.69%) and elevated resistance (+147.61%), consistent with membrane injury and impaired ion transport. Although xylem pathways dominated HCO₃⁻ transport, the phloem—despite greater sensitivity—showed an increased relative contribution under stress, supporting partial compensatory allocation. Metabolically, severe stress induced a shift toward a “low-metabolism, high-efficiency” strategy, prioritizing water conservation over carbon assimilation. Collectively, Bletilla striata adopts a dual strategy: maintaining functional stability (and modest enhancement) under environmentally relevant bicarbonate concentrations, while shifting to conservative resource-use under excessive stress. These adaptive mechanisms highlight B. striata’s potential for ecological restoration and sustainable cultivation in bicarbonate-rich karst environments.

1. Introduction

Karst regions play a unique role in the global carbon cycle, acting as crucial terrestrial carbon sinks. The karst carbon cycle involves the coupling of lithospheric, hydrospheric, and biospheric processes, where carbonate rock dissolution converts atmospheric and soil-derived CO₂ into bicarbonate (HCO₃⁻) in karst water systems [1]. This dissolved inorganic carbon is further re-assimilated by aquatic and terrestrial organisms through photosynthesis, forming a “carbonate–biological carbon pump” that drives long-term CO₂ sequestration in karst ecosystems [2]. However, global warming is diminishing nitrogen fixation in these regions, disrupting the synergistic relationship between vegetation growth and the carbon cycle and threatening the stability of carbon sinks [3]. Moreover, the high-bicarbonate conditions characteristic of karst soils impose physiological constraints on plants, further limiting their carbon sequestration efficiency [4]. Consequently, identifying karst-adapted plants and elucidating their adaptive mechanisms are critical for enhancing the carbon sink capacity of karst ecosystems [5].

Bletilla striata (Thunb.) Rchb.f. is a perennial orchid and traditional Chinese medicinal herb [6] that is widely cultivated. Its underground pseudobulbs are rich in bioactive compounds, including glucosides, hydrobenzyl, phyllanthin, quinones, dihydrophyllanthin, anthocyanins, steroids, triterpenoids, and phenolic acids [7]. These constituents confer diverse biological activities—immune regulation, anti-inflammatory, anti-tumor, anti-fibrotic, hemostatic, and gastroprotective effects [8,9]—endowing B. striata with substantial medicinal and economic value in pharmaceuticals, cosmetics, and fine chemicals [10]. Owing to its fleshy root system that anchors firmly in shallow soils and pseudobulbs capable of storing water and withstanding drought, B. striata is considered a promising medicinal and ecological restoration species for cultivation in karst rocky desertification areas [11]. Multi-generational cultivation and selective breeding by Wu Mingkai’s team—exemplified by the “saddle-shaped” cultivar Guiji No.1—have further enhanced its adaptability and produced plants with exceptional tolerance to karst conditions [12]. Despite these advances, the adaptive strategies of B. striata remain incompletely understood, particularly the physiological regulation of intracellular water metabolism and nutrient transport under varying HCO₃⁻ stress.

The effects of bicarbonate salts on plants display dual characteristics: they are both concentration-dependent and species-specific. At high concentrations, bicarbonate ions (HCO₃⁻) inhibit growth and induce chlorosis [13]. At high external pH, alkalinity itself can inhibit plant growth by altering rhizosphere chemistry and micronutrient availability. Elevated pH suppresses ferric-chelate reductase (FCR) activity and downregulates Fe acquisition genes such as FRO, IRT1, and H⁺-ATPase, thereby constraining Fe(III) reduction and root-to-shoot iron transport [14,15]. However, beyond the indirect effects of alkalinity, bicarbonate ions (HCO₃⁻) exert additional, direct physiological inhibition. Studies have shown that even when rhizosphere pH is buffered, HCO₃⁻ itself can directly inhibit root FCR activity, as demonstrated in the dwarfing rootstock quince A (Cydonia oblonga Mill.) [16]. In addition, HCO₃⁻ disrupts nitrogen metabolism by reducing nitrate reductase (NR) and glutamine synthetase (GS) activities [17,18], and compromises plasma membrane integrity by inducing K⁺ and NO₃⁻ efflux, promoting the accumulation of reactive oxygen species and organic acids, and collectively impairing cell division and root elongation [19,20].

In contrast to these inhibitory effects, low concentrations of HCO₃⁻ may exert beneficial or signaling roles independent of alkalinity [21]. In Arabidopsis, treatment with 0.5–3 mM sodium bicarbonate increased leaf number, improved fresh and dry weight, and reduced ion leakage [22]. Exposure to ~1 mM HCO₃⁻ enhanced nitrate assimilation, nitrogen translocation to shoots, and biomass accumulation [23] In drought-adapted species, HCO₃⁻ also functions as an auxiliary inorganic carbon source, replenishing photorespiratory CO₂ losses and reactivating photosynthesis by elevating PSII quantum yield (Fv/Fm) and stomatal conductance [24,25]. Mechanistically, HCO₃⁻ redirects glycolytic flux toward the pentose phosphate pathway (PPP) via glucose-6-phosphate dehydrogenase activation, increasing NADPH supply for antioxidant regeneration (e.g., glutathione) [26]. Simultaneously, it enhances phosphoenolpyruvate carboxylase (PEPC) activity, supporting carbon skeleton production for amino acid biosynthesis and cytosolic pH stabilization [18]. These direct and concentration-dependent responses depend on the systemic translocation of HCO₃⁻ through vascular tissues, particularly via the xylem and phloem pathways.

Bicarbonate (HCO₃⁻) undergoes systemic redistribution in plants through coordinated transport along both the xylem and phloem pathways [27]. Along the root–shoot axis, it is absorbed and carried apoplastically in the xylem, driven by the transpiration stream [28]. Radial exchange between the two vascular systems further enables inter-vascular transfer, thereby contributing to the dynamic regulation of carbon and water homeostasis [29]. In karst environments, where soil HCO₃⁻ concentrations are inherently high, clarifying HCO₃⁻ transport dynamics in xylem and phloem directly reveals how plants balance “utilizing HCO₃⁻ as a carbon source” and “avoiding HCO₃⁻-induced physiological toxicity”—a core mechanism underlying karst adaptation. In the phloem, HCO₃⁻ or its CO₂-derived equivalents are loaded into sieve elements via symplastic or apoplastic routes and delivered from sources to sinks by the pressure-flow mechanism [21,30]. Carbonic anhydrases accelerate the reversible conversion between CO₂ and HCO₃⁻, ensuring efficient uptake and long-distance redistribution [31]. Elucidating these vascular transport processes is therefore central to understanding the roles of bicarbonate in photosynthetic carbon assimilation, pH regulation, and stress adaptation across diverse plant taxa.

Plant electrophysiology has emerged as a powerful approach to probing rapid physiological responses and transport dynamics [32], and recent advancements in sensing and modeling have transformed electrical signals into computable phenotypic data, propelling the field into an era of systems science [33]. Traditional electrical sensors and patch-clamp techniques can capture transient water and nutrient states [34], but they fall short in resolving dynamic transport processes or linking bioelectrical signals with energy conversion and metabolic fluxes [35]. Recent methodological advances now allow the use of analysis of electrophysiological parameters—such as capacitance (IC), resistance (IR), and impedance (IZ)—to non-invasively characterize cellular water dynamics, nutrient transport, and energy conversion efficiency in real time [33]. The approach has proven effective across multiple contexts, including assessments of stress tolerance [36], early disease detection [37], and resource-use efficiency [38]. For example, measurements of leaf resistance and capacitance have quantified the drought adaptability of Broussonetia papyrifera (L.) L’Hér. ex Vent. and Morus alba L. [36]; electrophysiological profiling has clarified salt tolerance in mangroves [39]; and physiological traits in continuous-cropped peanut (Arachis hypogaea L.) under CSC LY05 + LGY06 treatment have been effectively assessed [40].

Based on previous evidence that bicarbonate (HCO₃⁻) can act as both a carbon source and a stressor in plants, we hypothesized that B. striata exhibits concentration-dependent electrophysiological and physiological responses to HCO₃⁻ stress. Specifically, low concentrations were expected to maintain or enhance photosynthetic efficiency and intracellular stability, whereas high concentrations would impair water and nutrient transport and alter membrane electrical properties. This study uses these techniques on B. striata “Gui Ji No. 1” to systematically examine growth, photosynthetic performance, intracellular water metabolism, and nutrient transport under varying bicarbonate stresses. The study aims to resolve the intrinsic links between electrophysiological traits, photosynthetic responses, and physiological regulation. Specifically, we address: (1) the effects of HCO₃⁻ on photosynthesis and chlorophyll content; (2) adjustments in intracellular water use and nutrient transport; (3) the fate and transport mechanisms of exogenous bicarbonate after incorporation; and (4) the basis of karst adaptability in B. striata. Ultimately, these findings illuminate the physiological strategies underpinning bicarbonate tolerance in karst ecosystems, offering theoretical insights and practical guidance for optimizing cultivation and selecting species for ecological restoration.

2. Materials and Methods

2.1. Plant Materials

The experimental material was the saddle-type B. striata (variety: Gui Ji No. 1), obtained from the Crop Variety Resources Institute of Guizhou Province (Guiyang, China). Using a completely randomized design, seedlings were allocated into 48 cultivation pots (One pot with one plant) with 30 cm spacing between pots to minimize microenvironmental effects (e.g., light and ventilation). Cultivation commenced on 20 November 2024. Uniform seedlings were selected, rinsed three times with distilled water, and transplanted into plastic pots (18.5 cm in diameter, 16.5 cm in height) containing 2 kg of a perlite-vermiculite substrate (2:1, v/v). The plants were cultivated in a controlled greenhouse at the Institute of Geochemistry, Chinese Academy of Sciences, under the following conditions: a light intensity of 500 μmol m⁻² s⁻¹, a 12 h photoperiod, an air temperature of 25 ± 1 °C, and a relative humidity of 60 ± 5%. During the initial two-week establishment period (20 November to 4 December 2024), seedlings were irrigated with a modified half-strength Hoagland’s solution (

Table A1. The modified composition of Hoagland nutrient solution.

Macroelement	Quantity of Matter (mM)
KNO3	6
NH4Cl	0.75
NH4H2PO4	0.25
Ca(NO3)2•4H2O	4
MgSO4•7H2O	2
A trace element
KCl	2
H3BO3	50
CuSO4•5H2O	0.2
ZnSO4•7H2O	4
MnSO4•4H2O	4
(NH4)6Mo7O24•4H2O	0.2
Carnallite
Fe(Na)EDTA	2

). Subsequently, starting on 5 December 2024, they were supplied with full-strength Hoagland’s solution for the remainder of the cultivation period. Since underground rhizome growth could not be monitored directly, a secondary selection was conducted on 5 January 2025. The screening criteria were as follows: (1) presence of three fully expanded functional leaves; (2) a shoot height of 8–10 cm and a pseudobulb diameter of ≥1 cm; and (3) healthy root systems without decay or pest damage. This final selection resulted in 16 pots (4 treatments × 4 replicates). Individuals exhibiting uniform above- and below-ground development were then transplanted and grown for an additional month until the three-leaf stage was consistently reached. Bicarbonate treatments were subsequently initiated, with each treatment consisting of four replicate pots (n = 4).

2.2. Bicarbonate Treatment and Concentration Maintenance

The experiment consisted of four treatment groups with varying bicarbonate concentrations. NaHCO₃ (AR grade, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was used as the source of bicarbonate. Specifically, the treatments were designated as follows: CK (0 mM NaHCO₃), T1 (5 mM NaHCO₃), T2 (10 mM NaHCO₃), and T3 (15 mM NaHCO₃). The nutrient solutions corresponding to each treatment were uniformly applied to the cultivation pots at a volume of 1.5 L per pot. The plants were cultivated under these treatments for a duration of 45 days (17 February–3 April 2025). To maintain a consistent bicarbonate concentration, the treatment solutions (CK–T3) were replaced daily at 14:00, with the volume kept constant at 1.5 L. Each treatment group was established with four biological replicates.

2.3. Chlorophyll Content and Biomass

Chlorophyll content of B. striata leaves was determined on 3 April 2025 with a chlorophyll meter (TYS-4N, Jinkelida, Beijing, China). After calibration with no sample, six measurements were taken per leaf—two each at the tip, middle, and base. After photosynthetic and electrophysiological measurements, plants were harvested, washed free of vermiculite, and blotted dry. Samples were separated into roots, stems, and leaves, and the fresh weight of each organ was recorded to calculate root, stem, leaf, and total biomass.

2.4. Photosynthesis

Following the treatment period, photosynthetic parameters of B. striata leaves were measured between 09:00 and 11:00 on 3 April 2025 using a portable photosynthesis system (LI-6400XT, LI-COR, Lincoln, NE, USA). The measurement protocol was strictly standardized as follows: reference CO₂ concentration was set to 400 μmol·mol⁻¹, photosynthetically active radiation (PAR) to 500 μmol·m⁻²·s⁻¹, block temperature (controlling leaf temperature) to 25 °C, and airflow rate to 500 μmol·s⁻¹ with the leaf fan set to “Fast” mode. Prior to measurement, the instrument was preheated for 30 min, calibrated via IRGA zero adjustment (for CO₂ and H₂O), and validated for stable gas exchange (CO₂ fluctuation < 2 μmol·mol⁻¹ under scrub mode, flow rate fluctuation ± 2 μmol·s⁻¹). Measurements included net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and transpiration rate (E). Instantaneous water-use efficiency (WUE, %) was calculated according to the following formula [41]:

$$\mathrm{W}\mathrm{U}\mathrm{E}={\displaystyle \frac{P_{\mathrm{n}}}{E}}$$

(1)

In Equation (1), P_n and E were measured in μmol CO₂ m⁻² s⁻¹ and mmol H₂O m⁻² s⁻¹, respectively; thus, WUE is expressed in μmol CO₂ mmol⁻¹ H₂O.

2.5. Intrinsic Electrophysiological Parameters of B. striata Leaves

At the end of the bicarbonate treatment period (3 April 2025), plants exhibiting uniform growth and health across treatments were selected for electrophysiological analysis. Real-time measurements of leaf electrical properties were obtained using a parallel-plate capacitor connected to an LCR meter (Model LCR-6100, Good Will Instrument Co., Ltd., Suzhou, China) during the same time window as the photosynthetic measurements (09:00–11:00). Because vascular bundles are densely distributed and highly lignified, direct electrode contact with these tissues can markedly increase local impedance, thereby compromising measurement accuracy. To minimize this systematic error, measurement points were taken near the leaf tip. Data were collected from the first, second, and third leaves of each plant. During measurements, leaves were clamped between the parallel plates under a test voltage of 1.5 V and a frequency of 3 kHz. The capacitance sensors were operated in parallel mode through the LCR meter. Physiological resistance (R), impedance (Z), and capacitance (C) were recorded under varying clamping forces (F), with 15–20 readings collected per force. Fifteen datasets were retained for subsequent calculations to ensure robustness. Leaf reactance (Xc) and inductive reactance (XL) were first derived from the primary measurements using Equations (2) and (3). Based on these values and following previous work [35], the relationships between clamping force (F) and the electrical parameters (C, R, Z, Xc, XL) were then characterized by the fitting equations in Equations (4)–(8). Finally, the intrinsic electrical parameters at zero clamping force (F = 0 N)—namely the inherent conduction resistance (IR), intrinsic impedance (IZ), intrinsic capacitive reactance (IXc), intrinsic inductive reactance (IXL), and intrinsic capacitance (ICP)—were determined by evaluating the fitted models in Equations (9)–(13).

$$\mathrm{X}\mathrm{c}={\displaystyle \frac{1}{2\mathsf{\pi }\mathrm{f}\mathrm{C}}}$$

(2)

$${\displaystyle \frac{1}{-\mathrm{X}\mathrm{c}}}={\displaystyle \frac{1}{\mathrm{Z}}}-{\displaystyle \frac{1}{\mathrm{R}}}-{\displaystyle \frac{1}{\mathrm{Xc}}}$$

(3)

$$\mathrm{R}={\mathrm{y}}_1+{ \mathrm{a}}_1{\mathrm{e}}^{-{\mathrm{b}}_1\mathrm{F}}$$

(4)

$$\mathrm{Z}={ \mathrm{y}}_2+{\mathrm{a}}_2{\mathrm{e}}^{{-\mathrm{b}}_2\mathrm{F}}$$

(5)

$$\mathrm{Xc}={\mathrm{y}}_3+{\mathrm{a}}_3{\mathrm{e}}^{{-\mathrm{b}}_3\mathrm{F}}$$

(6)

$$\mathrm{X}\mathrm{L}={\mathrm{y}}_4+{\mathrm{a}}_4{\mathrm{e}}^{{-\mathrm{b}}_4\mathrm{F}}$$

(7)

$$\mathrm{C}={\mathrm{y}}_0+\mathrm{a}\mathrm{F}$$

(8)

$$\mathrm{IR}={\mathrm{y}}_1+{\mathrm{a}}_1\mathrm{F}$$

(9)

$$\mathrm{IZ}={\mathrm{y}}_2+{\mathrm{a}}_2\mathrm{F}$$

(10)

$$\mathrm{IXc}={\mathrm{y}}_3+{\mathrm{a}}_3\mathrm{F}$$

(11)

$$\mathrm{I}\mathrm{XL}={\mathrm{y}}_4+{\mathrm{a}}_4\mathrm{F}$$

(12)

$$\mathrm{ICP}={\displaystyle \frac{1}{2\mathsf{\pi } \mathrm{fIXc}}}$$

(13)

In Equation (2), π = 3.1416 and f = 3000 Hz.

2.6. Intracellular Water Use Dynamics

Intracellular water use in leaves was assessed through several parameters. Intracellular water-holding capacity (IWHC) was calculated using Equation (14), while the effective leaf thickness (d) was determined with Equation (15). Intracellular water-use efficiency (IWUE), intracellular water-holding time (IWHT), and water transfer rate (WTR) were calculated using Equations (16)–(18):

$$\mathrm{IWHC} = \sqrt{{\left(\mathrm{ICP}\right)}^3}$$

(14)

$$\mathrm{d}={\displaystyle \frac{{\mathrm{U}}^2\mathrm{a}}{2}}$$

(15)

$$\mathrm{IWUE}={\displaystyle \frac{\mathrm{d}}{\mathrm{IWHC}}}$$

(16)

$$\mathrm{IWHT}=\mathrm{ICP}\times \mathrm{IZ}$$

(17)

$$\mathrm{WTR}={\displaystyle \frac{\mathrm{IWHC}}{\mathrm{IWHT}}}$$

(18)

2.7. Characterization of Nutrient Translocation Capacity

Nutrient flux per unit area (UNF) was calculated using Equation (19). Because nutrients are water-soluble, WTR and the nutrient translocation rate (NTR) were treated as equivalent, as defined in Equation (20). Nutrient translocation capacity (NTC), active transport flow of nutrient (UAF), and nutrient active translocation capacity (NAC) were determined according to Equations (21)–(23):

$$\mathrm{UNF} = {\displaystyle \frac{\mathrm{I}\mathrm{R}}{\mathrm{IXc}}} + {\displaystyle \frac{\mathrm{I}\mathrm{R}}{\mathrm{I}\mathrm{XL}}}$$

(19)

$$\mathrm{NTR}=\mathrm{WTR}$$

(20)

$$\mathrm{N}\mathrm{T}\mathrm{C}=\mathrm{U}\mathrm{N}\mathrm{F}\times \mathrm{N}\mathrm{T}\mathrm{R}$$

(21)

$$\mathrm{U}\mathrm{A}\mathrm{F}={\displaystyle \frac{\mathrm{IXc}}{\mathrm{I}{\mathrm{X}}_{\mathrm{L}}}}$$

(22)

$$\mathrm{NAC}=\mathrm{UAF}\times \mathrm{NTR}$$

(23)

2.8. Metabolic Activity

Metabolic activity correlates with bioelectric current, with higher currents indicating more complete metabolism. Under a parallel circuit configuration where voltage (U) is constant, bioelectric currents were defined as follows: resistive bioelectric current (${\mathrm{I}}_{\mathrm{I}\mathrm{R}}={\displaystyle \frac{\mathrm{U}}{\mathrm{I}\mathrm{R}}}$), impedance-based bioelectric current (${\mathrm{I}}_{\mathrm{I}\mathrm{Z}}={\displaystyle \frac{\mathrm{U}}{\mathrm{I}\mathrm{Z}}}$), capacitive reactance-based bioelectric current (${\mathrm{I}}_{\mathrm{X}\mathrm{c}}={\displaystyle \frac{\mathrm{U}}{\mathrm{IX}\mathrm{c}}}$), and inductive reactance-based bioelectric current (${\mathrm{I}}_{\mathrm{X}\mathrm{L}}={\displaystyle \frac{\mathrm{U}}{\mathrm{IX}\mathrm{L}}}$). The metabolic flow (MF) of leaf cells was calculated using Equation (24):

$$\mathrm{MF} ={\displaystyle \frac{{10}^5}{ \mathrm{IR}\times \mathrm{IZ}\times \mathrm{IXc}\times \mathrm{IX}\mathrm{L}}}$$

(24)

Cell metabolic intensity (MS), leaf metabolic rate (MR), relative metabolic activity (MA), and the growth comprehensive score (GCS) were quantified using Equations (25)–(28):

$$\mathrm{MS} = \ln \left(\mathrm{MF}\right)$$

(25)

$$\mathrm{MR}=\mathrm{NTR}\times \mathrm{NAC}$$

(26)

$$\mathrm{MA}=\sqrt[6]{\mathrm{MF}\times \mathrm{MR}}$$

(27)

$$\mathrm{GCS}=\sqrt[4]{\mathrm{IWHC}\times \mathrm{IWHT}\times \mathrm{NTC}\times \mathrm{MA}}$$

(28)

2.9. Inherent Conduction Capacity and Inherent Conduction Resistance

Leaf physiological current reflects the conductive performance of polar substances, specifically leaf hydraulic conductance. Because impedance (Z) is inversely proportional to the transport capacity of dielectric substances, the inherent conduction capacity can be derived. According to Ohm’s law, I_Z = U/Z, where U is the applied voltage (1.5 V) and I_Z is the physiological current. Combining this with the clamping force–impedance relationship yields [42]:

$${\mathrm{I}}_{\mathrm{Z}}={\displaystyle \frac{\mathrm{U}}{{\mathrm{y}}_2+{{\mathrm{a}}_2\mathrm{e}}^{-{\mathrm{b}}_2\mathrm{F}}}}$$

(29)

Differentiation of Equation (29) gives:

$${\mathrm{I}}_{\mathrm{Z}}^{\prime }={\displaystyle \frac{\mathrm{U}{\mathrm{b}}_2{\mathrm{a}}_2}{({\mathrm{y}}_2+{{\mathrm{a}}_2\mathrm{e}}^{-{\mathrm{b}}_2\mathrm{F}})({\mathrm{y}}_2+{{\mathrm{a}}_2\mathrm{e}}^{-{\mathrm{b}}_2\mathrm{F}})}}$$

(30)

$${\mathrm{I}\mathrm{C}\mathrm{C}}_{\mathrm{Z}}={\displaystyle \frac{\mathrm{U}{\mathrm{b}}_2{\mathrm{a}}_2}{{(\mathrm{y}}_2+{\mathrm{a}}_2){(\mathrm{y}}_2+{\mathrm{a}}_2)}}$$

(31)

$${\mathrm{I}\mathrm{C}\mathrm{C}}_{\mathrm{R}}={\displaystyle \frac{\mathrm{U}{\mathrm{b}}_1{\mathrm{a}}_1}{{(\mathrm{y}}_1+{\mathrm{a}}_1){(\mathrm{y}}_1+{\mathrm{a}}_1)}}$$

(32)

$${\mathrm{I}\mathrm{C}\mathrm{C}}_{\mathrm{X}\mathrm{c}}={\displaystyle \frac{\mathrm{U}{\mathrm{b}}_3{\mathrm{a}}_3}{{(\mathrm{y}}_3+{\mathrm{a}}_3){(\mathrm{y}}_3+{\mathrm{a}}_3)}}$$

(33)

$${\mathrm{I}\mathrm{C}\mathrm{C}}_{\mathrm{X}\mathrm{L}}={\displaystyle \frac{\mathrm{U}{\mathrm{b}}_4{\mathrm{a}}_4}{{(\mathrm{y}}_4+{\mathrm{a}}_4){(\mathrm{y}}_4+{\mathrm{a}}_4)}}$$

(34)

The physiological impedance thus inversely reflects the conductive performance of polar substances, i.e., leaf hydraulic conductance, and defines impedance-based conduction resistance:

$$\mathrm{Z}\prime =-{\mathrm{b}}_2{\mathrm{a}}_2{\mathrm{e}}^{-{\mathrm{b}}_2\mathrm{F}}$$

(35)

When F = 0, the inherent transport resistance is expressed as:

$${\mathrm{I}\mathrm{C}\mathrm{R}}_{\mathrm{Z}}=-{\mathrm{b}}_2{\mathrm{a}}_2$$

(36)

$${\mathrm{I}\mathrm{C}\mathrm{R}}_{\mathrm{R}}=-{\mathrm{b}}_1{\mathrm{a}}_1$$

(37)

$${\mathrm{I}\mathrm{C}\mathrm{R}}_{\mathrm{X}\mathrm{c}}=-{\mathrm{b}}_3{\mathrm{a}}_3$$

(38)

$${\mathrm{I}\mathrm{C}\mathrm{R}}_{\mathrm{X}\mathrm{L}}=-{\mathrm{b}}_4{\mathrm{a}}_4$$

(39)

2.10. Statistical Analysis

Statistical analyses were performed using a one-way analysis of variance (ANOVA). Post hoc comparisons among treatments were conducted with Duncan’s multiple range test. Relationships between variables were further examined using Pearson correlation analysis. Statistical significance was set at p ≤ 0.05. While data are expressed as mean ± standard deviation (SD) based on four biological replicates (n = 4) in tables, the 95% confidence intervals (95% CI) were also calculated to assess variability and the precision of mean estimates.

3. Results

3.1. Influence of HCO₃⁻ on Growth Characteristics and Chlorophyll Content of B. striata

As shown in

Table 1. Biomass of roots, stems, and leaves of B. striata.

Treatment	CK	T1	T2	T3
Root (FW, g/Plant)	3.59 ± 0.40 a	3.44 ± 0.46 a	2.69 ± 0.22 b	2.39 ± 0.32 b
Stem (FW, g/Plant)	6.12 ± 0.44 a	5.91 ± 0.85 ab	5.41 ± 0.66 bc	4.83 ± 0.72 c
Leaves (FW, g/Plant)	2.64 ± 0.47 a	2.59 ± 0.30 a	2.17 ± 0.51 b	1.71 ± 0.33 c
Total Biomass (FW, g/Plant)	12.35 ± 0.41 a	11.94 ± 0.41 a	10.27 ± 0.25 b	8.94 ± 0.71 c

Data are presented as mean ± SD (n = 4). Different superscript letters within a column indicate significant differences (p < 0.05) by Duncan’s multiple range test.

, B. striata biomass declined progressively with increasing NaHCO₃ concentrations (0–15 mM). Control plants (CK) displayed the highest root, stem, and leaf biomass, resulting in maximal total biomass. At 5 mM (T1), slight reductions were observed in all organs, but these differences were not significant compared with CK. In contrast, severe stress at 15 mM (T3) reduced total biomass by 27.61%, markedly suppressing root, stem, and leaf growth, with roots and leaves exhibiting the sharpest declines. Chlorophyll content was unaffected at T1 but declined significantly under moderate stress (T2, 10 mM) and further dropped to 23.28% below CK at T3, representing reductions of 3.02%, 9.20% and 23.28% at T1–T3, respectively.

3.2. Influence of HCO₃⁻ on Photosynthetic Characteristics of B. striata

Significant differences in P_n, C_i, and WUE were observed among treatments (p < 0.05). Relative to CK, P_n decreased by 7.63%, 21.36%, and 51.32% under T1, T2, and T3, respectively. G_s and E showed similar patterns: no significant differences between CK and T1, a sharp decline at T2, and the lowest values at T3, with reductions of 65.37% and 62.46%, respectively. C_i varied inversely with WUE, ranking T1 > CK > T2 > T3, whereas WUE followed the opposite order, T3 > T2 > CK > T1.

3.3. Influence of HCO₃⁻ on Electrophysiological Parameters of B. striata

The electrophysiological parameters of B. striata leaves varied significantly with HCO₃⁻ concentration. Intrinsic capacitance (IC) declined markedly (p < 0.05), falling from 44.38 ± 3.16 in CK (0 mM) to 22.77 ± 1.05 in T3 (15 mM), a reduction of ~48.69%. By contrast, IR, IZ, IXc, and IXL increased significantly with rising HCO₃⁻ (p < 0.05), with respective increases of 147.61%, 84.68%, 94.17%, and 152.64% from CK to T3. No significant differences were detected between CK and T1 (5 mM) for any parameter (p > 0.05), whereas values rose sharply from T2 (10 mM) and peaked at T3.

3.4. Influence of HCO₃⁻ on Intracellular Water Metabolism Capacity of B. striata

Significant differences in relative IWHC were observed among treatments (p < 0.05). From CK (0 mM) to T1 (5 mM), IWHC declined gradually by 3.57%, a change that was not significant (p > 0.05). At higher NaHCO₃ concentrations, reductions became pronounced: IWHC decreased by 18.14% at T2 (10 mM) and fell sharply at T3 (15 mM), reaching the lowest value with a 35.90% reduction relative to CK (p < 0.05). By contrast, IWUE increased significantly with rising NaHCO₃ (p < 0.05), being lowest in CK, modestly elevated in T1 (+0.23%) and T2 (+19.29%), and peaking in T3 with a 45.83% increase over CK. IWHT showed no significant differences across treatments; although CK displayed slightly greater dispersion and a higher mean, the variation was not statistically significant (p > 0.05).

3.5. Influence of HCO₃⁻ on Nutrient Transport Dynamics of B. striata

The parameters displayed distinct treatment-dependent patterns. UNF followed the order T2 > T3 > CK > T1, with T2 reaching the maximum. Although T3 was 13% lower than T2, it remained significantly higher than CK and T1. For NTR and WTR, the highest values occurred in T1 (+2.29% vs. CK, nonsignificant), while both T2 and T3 were significantly reduced relative to CK. NTC ranked T2 > T1 > CK > T3: T1 was only marginally higher than CK (+1.55%, nonsignificant), T2 recorded the maximum (+18.36% vs. CK), and T3 was the lowest, with a significant reduction of 22.26%. UAF peaked in CK, declined slightly in T1 (−8.08%, nonsignificant), and reached the minimum in T2 (−36.37% vs. CK). T3 partially rebounded (+21.64% vs. T2) but remained 22.60% below CK. NAC remained stable in CK and T1 but declined markedly in T2 and T3 (−45.42% and −47.87% vs. CK).

3.6. Influence of HCO₃⁻ on Metabolic Indices of B. striata

Under bicarbonate stress, the mesophyll structure (MS), mesophyll ratio (MR), and mesophyll area (MA) of B. striata leaves declined in a concentration-dependent manner. MS decreased slightly in T1 (−3.04% vs. CK, p > 0.05), but declined significantly in T2 and T3 (−24.45% and −37.83%, p < 0.05). MR showed a similar trend, with reductions of 4.18%, 53.24%, and 64.97% in T1, T2, and T3, respectively; T1 did not differ from CK (p > 0.05). For MA, decreases were 4.88%, 37.03%, and 50.00% in T1, T2, and T3, respectively. All three indices reached their lowest levels at T3, which differed significantly from all other treatments (p < 0.05), while T1 remained statistically indistinguishable from CK.

3.7. Influence of HCO₃⁻ on Inherent Conduction Capacity and Inherent Conduction Resistance of B. striata

Inherent conduction capacity declined progressively with increasing bicarbonate concentration, whereas inherent conduction resistance increased. Across models, CK and T1 showed no significant differences, but sharp changes emerged from T2 onwards. In the conductance models (ICC_R, ICC_XL, ICC_Z, ICC_Xc), values fell significantly at T2 and reached reductions of ~32–38% at T3. In contrast, inherent conduction resistance rose steeply under stress: in the R–X_L model it increased more than threefold at T3, while in the R–X_C model it rose over onefold, with both showing significant elevations from T2. Consistently, the ratio of ICC_Xc to ICC_XL declined from 453.66% in CK to 401.16% at T2 before partially recovering at T3 (422.18%), while the ratio of ICR_XC to ICR_XL decreased from 35.01% in CK to a minimum of 12.30% at T2, rebounding to 19.01% at T3.

4. Discussion

4.1. The Effects of Bicarbonate Ions on the Growth and Photosynthesis of B. striata

Bicarbonate ions (HCO₃⁻) exerted a concentration-dependent suppression of growth and photosynthetic performance in B. striata. At 15 mM NaHCO₃, total biomass, chlorophyll content, and P_n decreased, respectively (Table 1, Figure 1a,b and Figure 2a). However, under mild exposure (T1, 5 mM), most physiological parameters remained statistically indistinguishable from the control, indicating a tolerance threshold and reflecting the adaptive capacity of B. striata to low bicarbonate levels typical of karst soils (Figure 2).

Under moderate to severe stress (T2 and T3), photosynthetic inhibition involved both stomatal and non-stomatal constraints. Declines in stomatal conductance (G_S) and transpiration (E) restricted CO₂ supply, reducing P_n (Figure 2), a stomatal-dominated limitation pattern resembling drought- and salinity-induced responses in which stomatal closure conserves water at the cost of carbon assimilation [43,44]. At higher bicarbonate concentrations, the sharp drop in intercellular CO₂ (Ci) indicated that stomatal restriction outweighed biochemical capacity, whereas the biphasic C_i response—initial elevation followed by suppression—may signal early photochemical impairment. Chlorophyll loss (Figure 1b) pointed to non-stomatal inhibition, likely from disrupted Fe and Mg homeostasis, both critical cofactors for chlorophyll biosynthesis and pigment–protein stability [45]. Comparable mechanisms have been reported in quince rootstock (Cydonia oblonga Mill.), where HCO₃⁻ suppressed ferric-chelate reductase activity, causing iron-deficiency chlorosis 1316. Structural and biochemical evidence from Brassica napus L. and Populus euphratica Oliv. further shows that excess bicarbonate damages chloroplast ultrastructure, suppresses Rubisco activity, and reduces electron transport efficiency [46,47]. Notably, water-use efficiency (WUE) increased under T3 owing to reduced transpiration. Although this resembles the water-conserving responses typical of karst-adapted plants, it more likely reflects a passive outcome of stomatal closure than genuine enhancement of carbon assimilation, underscoring a trade-off between water conservation and photosynthetic productivity [48,49].

4.2. Mechanism of HCO₃⁻ on Intracellular Water, Nutrient Translocation, and Metabolic Processes in B. striata

4.2.1. Electrophysiological Responses of B. striata to HCO₃⁻ Stress

Electrophysiological profiling revealed concentration-dependent shifts in the membrane electrical properties of B. striata under bicarbonate stress (

Table 2. Intrinsic electrophysiological traits of B. striata leaves across bicarbonate levels.

Treatment	IC (pF)	IR (MΩ)	IZ (MΩ)	IXC (MΩ)	IXL (MΩ)
CK (0 mM)	44.38 ± 3.16 a	4.18 ± 0.43 c	1.24 ± 0.05 c	1.20 ± 0.08 c	4.35 ± 0.48 b
T1 (5 mM)	42.03 ± 2.72 a	4.45 ± 0.38 c	1.24 ± 0.12 c	1.27 ± 0.08 c	4.98 ± 0.14 b
T2 (10 mM)	32.90 ± 2.30 b	8.53 ± 0.57 b	1.60 ± 0.11 b	1.62 ± 0.11 b	9.20 ± 0.87 a
T3 (15 mM)	22.77 ± 1.05 c	10.35 ± 0.81 a	2.29 ± 0.09 a	2.33 ± 0.11 a	10.99 ± 1.20 a

Data are presented as mean ± SD (n = 4). Different superscript letters within a column indicate significant differences (p < 0.05) by Duncan’s multiple range test.

). At low levels (5 mM NaHCO₃), intrinsic conductance (IC) and resistance (IR) were statistically unchanged, indicating preserved dielectric integrity and structural stability. By contrast, moderate to severe stress (T2–T3) reduced IC while elevating IR and impedance (IZ), consistent with restricted ion mobility and reduced membrane permeability [50]. Concurrent increases in IX_C and IX_L further pointed to impaired electrogenic pump activity, particularly H⁺-ATPases [51], which sustain transmembrane potential and drive secondary nutrient transport. These impedance patterns mirror universal stress signatures reported across plant taxa, where declines in capacitance coupled with elevated resistance mark early dielectric disruption and compromised ion transport capacity [32,39].

4.2.2. Coupled Mechanisms of Water, Nutrient, and Metabolic Regulation in B. striata Under HCO₃⁻ Stress

Bicarbonate stress induced a coordinated reconfiguration of intracellular water status, nutrient transport, and metabolic activity in B. striata, indicative of an integrated adaptive strategy to carbonate-driven osmotic and ionic stress. Under severe exposure (15 mM HCO₃⁻), intracellular water-holding capacity (IWHC) declined, reflecting substantial cellular dehydration, whereas intracellular water-use efficiency (IWUE) rose, indicating a compensatory shift that maximizes metabolic return per unit water retained (Figure 3a,b). Intriguingly, intracellular water-holding time (IWHT) remained stable (~52 s), implying that B. striata prioritizes water-use efficiency rather than prolonging retention to sustain basal metabolism (Figure 3c). This “efficiency-first” strategy parallels responses in Coix lacryma-jobi L., another karst-adapted species that enhances IWUE under carbonate-induced drought [52]. By contrast, water transfer rate (WTR) declined markedly, pointing to a regulatory threshold beyond which aquaporin gating and phosphorylation-dependent control restrict transmembrane flux, elevating apoplastic resistance [53]. Physiologically, the negative correlation between IWUE and photosynthetic rate underscores a trade-off in which carbon assimilation is downregulated to conserve water, consistent with ABA-mediated stomatal closure as a dominant photosynthetic limitation under stress [54].

Nutrient transport pathways exhibited clear concentration-dependent adjustments. At 5 mM HCO₃⁻, both NAC and UNF remained statistically unchanged, indicating preserved proton-driven transport (Figure 4a,e). By contrast, under severe stress (15 mM HCO₃⁻), IC and NAC declined, while IR, UNF, and IWUE rose, indicating a possible shift from proton-driven active uptake toward concentration-driven and exchange-based uptake [55]. Concurrently, NTR and NTC decreased, respectively, reflecting impaired systemic allocation (Figure 4b,c), while the reduction in UAF further indicated the downregulation of energy-intensive transport (Figure 4d). These patterns can be inferred to suggest that bicarbonate stress reprograms nutrient fluxes in part through enhanced organic acid metabolism and exudation under alkaline conditions, which may buffer carbonate ions and improve nutrient-use efficiency [52,56]. Metabolic regulation was tightly coupled to these water–nutrient adjustments. At 15 mM HCO₃⁻, MA declined, accompanied by a marked rise in MS, reflecting an overall reduction in respiratory and biosynthetic activity (

Table 3. Metabolic storage (MS), metabolic rate (MR), metabolic adaptability (MA), and glycogen consumption speed (GCS).

Treatment	MS	MR	MA	GCS
CK (0 mM)	8.22 ± 0.18 a	144.59 ± 20.54 a	9.02 ± 0.35 a	89.09 ± 4.26 a
T1 (5 mM)	7.97 ± 0.24 a	138.55 ± 12.81 a	8.58 ± 0.47 a	87.2 ± 4.36 a
T2 (10 mM)	6.21 ± 0.30 b	67.61 ± 7.69 b	5.68 ± 0.39 b	78.04 ± 5.20 b
T3 (15 mM)	5.11 ± 0.14 c	50.65 ± 5.29 c	4.51 ± 0.18 c	62.89 ± 3.18 c

Data are presented as mean ± SD (n = 4). Different superscript letters within a column indicate significant differences (p < 0.05) by Duncan’s multiple range test.

). Nevertheless, the concurrent increases in IWUE and WUE highlight a strategic shift toward a “low-metabolism, high-water-efficiency” survival mode [57,58].

4.3. Xylem-Dominated Transport of Bicarbonate in B. striata, Phloem Exhibits Greater Sensitivity but an Increased Contribution to HCO₃⁻ Translocation

This study indicates that bicarbonate transport in B. striata is primarily xylem-driven, with the phloem exhibiting greater sensitivity to stress. The ratio of xylem to phloem conductance (ICC_Xc/ICC_XL) remained consistently around fourfold higher, whereas the corresponding resistance ratio (ICR_Xc/ICR_XL) was consistently below one (Figure 5a). This indicates that the xylem possessed superior transport efficiency, characterized by higher conductance and lower resistance relative to the phloem. Even though rising HCO₃⁻ concentrations reduced relative xylem conductance (rICC_Xc) and elevated resistance (rICR_Xc), the xylem maintained a distinct advantage (

Table 4. Inherent and relative conduction capacities and resistances of B. striata under bicarbonate stress. Parameters include inherent conduction capacity based on Xc/inherent conduction capacity based on XL (ICC_Xc/ICC_XL); Inherent conduction resistance based on Xc/inherent conduction resistance based on XL (ICR_Xc/ICR_XL); relative inherent conduction capacity (rICC_Xc, rICC_XL); and relative inherent conduction resistance (rICR_Xc, rICR_XL). rICC and rICR are ratios relative to CK (CK = 1.00). ICC_Xc/ICC_XL and ICR_Xc/ICR_XL are dimensionless xylem:phloem conductance/resistance ratios.

Treatment	CK	T1	T2	T3
ICCXc/ICCXL	453.66%	432.26%	401.16%	422.18%
ICRXc/ICRXL	35.01%	25.32%	12.30%	19.01%
rICCXc	1	94.33%	73.34%	62.91%
rICCXL	1	99.00%	82.93%	67.61%
rICRXc	1	104.69%	132.29%	235.85%
rICRXL	1	144.78%	376.49%	434.30%

), reflecting its reliance on bulk flow and apoplastic continuity, which are less dependent on energy coupling. Mechanistically, the decline in xylem conductance can be attributed to apoplastic alkalinization, which raises vessel wall pH and suppresses H⁺-pump activity [28], alongside reduced membrane capacitance, aquaporin gating-induced hydraulic uncoupling 53, and pit membrane charge screening that restricts coordinated water–ion flow and elevates ICR_Xc [29]. In addition, Ca²⁺–HCO₃⁻ interactions alter vessel wall electrochemical properties and porosity [59], further compounding transport resistance. Collectively, these processes account for the progressive decline in xylem conductance under bicarbonate stress.

In contrast, the phloem exhibited a much steeper decline in transport capacity, with rICR_XL rising more than fourfold and rICC_XL falling more sharply than xylem values (Figure 6). This heightened sensitivity reflects the strong dependence of phloem transport on proton motive force and symplastic connectivity 2730. Concurrently, alkaline stress induces callose deposition at plasmodesmata, restricting symplastic continuity between companion cells and sieve elements, while disruption of ionic homeostasis further compromises sieve plate conductivity [60]. The imbalance between xylem and phloem is most pronounced at intermediate bicarbonate levels (10 mM), when bulk xylem flux remains largely intact but phloem energy coupling has already collapsed, creating a physiological mismatch between resource supply and distribution. The partial recovery of ratios observed at higher concentrations, even amidst elevated phloem resistance, points to the existence of a potential recovery mechanism [61].

4.4. Karst Adaptability and Stress-Enhanced Quality Formation in Saddle-Type B. striata

Saddle-type B. striata exhibits remarkable adaptability to bicarbonate-rich karst environments through an integrated suite of buffering, water-use optimization, and metabolic reprogramming strategies. At environmentally relevant concentrations (3–7 mM HCO₃⁻), the species maintains photosynthetic stability and nutrient transport [21,27], reflecting robust physiological buffering. Under higher stress, coordinated adjustments in water use, nutrient allocation, and metabolism support survival via a “low-metabolism, high-efficiency” strategy, highlighting a trade-off between growth and resource conservation consistent with other karst-tolerant taxa [29,57,58]. Future work will translate these electrical signatures into mass-flow budgets by combining ¹³C-labelled NaHCO₃ pulse-chase, xylem sap micro-sampling. Such direct flux measurements will confirm the xylem-dominated HCO₃⁻ transport and quantify phloem compensatory contribution under escalating stress. Although secondary metabolism was not quantified here, downregulation of primary metabolism may create conditions favorable for secondary metabolite accumulation, supporting the concept of “stress-induced quality formation” [62]. Collectively, these adaptive responses enhance ecological resilience and suggest agronomic potential for optimizing medicinal compound yields in karst cultivation systems.

5. Conclusions

This study elucidates the concentration-dependent physiological and electrophysiological responses of B. striata to bicarbonate stress, providing mechanistic insights into its adaptability to karst environments. Under low bicarbonate exposure (5 mM), B. striata maintained stable photosynthetic function and metabolic activity, reflecting robust homeostatic regulation. At moderate bicarbonate concentration (10 mM), the species exhibited an adaptive reorganization of transport and metabolic pathways, characterized by enhanced nutrient flux and improved intracellular water-use efficiency, which together sustained physiological stability under moderate stress. By contrast, exposure to high bicarbonate levels (15 mM) resulted in pronounced physiological impairment, including reductions in biomass and photosynthetic capacity, accompanied by electrophysiological evidence of decreased inherent conductance and increased resistance, indicating xylem dysfunction and membrane instability. Despite these constraints, a modest compensatory response was observed in phloem-mediated transport, suggesting limited but measurable resilience in vascular regulation. Collectively, these results define a practical bicarbonate tolerance threshold for B. striata and demonstrate the potential of plant electrophysiological indicators as effective tools for evaluating vascular responses to carbonate stress. This study provides a scientific basis for the selection and cultivation of karst-adapted species and supports ecological restoration and sustainable management of bicarbonate-rich environments.