Effects of High-Temperature Stress on Physiological Characteristics and Propagation Potential of Pholidota chinensis Seedlings

1. Introduction

Pholidota chinensis (Orchidaceae) is a perennial epiphytic herb, widely distributed in southern China and Southeast Asia[1]. It possesses medicinal, ornamental, and ecological value[2,3,4,5]. P. chinensis also plays a significant role in maintaining the diversity and stability of the forest understory microecosystem[6]. However, owing to long-term overharvesting and biological characteristics such as tiny seeds, including minute seeds, low natural germination, and dependence on symbiotic fungi, the natural reproduction rate of P. chinensis is extremely low, and its wild resources have continued to decline. It is currently listed as a vulnerable or endangered species. Therefore, developing efficient and sustainable artificial propagation techniques is urgently needed to protect its wild germplasm resources, meet market demand, and achieve sustainable utilization[7,8].

Aseptic sowing technology, as an efficient means of seed-based in vitro propagation, serves as an important approach for the conservation and commercial production of rare and endangered orchids. By establishing a stable in vitro seed germination and seedling development system, large numbers of high-quality, disease-free seedlings with consistent genetic traits can be obtained within a short period, thereby substantially reducing dependence on wild resources. However, extreme high temperatures resulting from global climate change pose a serious threat to the production of aseptically sown seedlings, particularly during the acclimatization and transplantation stages[9,10]. During these stages, seedlings must gradually adapt from an in vitro environment characterized by high humidity, low light, and sufficient nutrients to external conditions. High temperature can easily aggravate water deficit, disrupt the stability of the photosynthetic system, and induce physiological and metabolic disorders, thereby reducing transplantation survival. High-temperature stress has become a key abiotic factor limiting seed germination, seedling survival, and the overall efficiency of aseptic sowing systems. Although some studies have investigated high-temperature stress responses in other orchid species, a systematic understanding of the relationship between physiological responses and propagation potential in P. chinensis seedlings remains lacking[11]. Re-generation capacity is an essential component of propagation potential. However, how this trait is impacted by heat stress has often been overlooked[12].

The thermolability of photosystem II (PSII) provides a plausible physiological link between heat stress and subsequent regeneration failure. A decline in Fv/Fm is among the earliest detectable responses to supraoptimal temperatures, often preceding visible injury (Berry and Björkman, 1980)[13]. Reduced Fv/Fm reflects impaired PSII photochemical efficiency caused by damage to the oxygen-evolving complex, electron transport chain, thylakoid membranes, and reaction centers. Chlorophyll fluorescence is therefore widely used as a rapid and non-destructive indicator of plant stress (Maxwell and Johnson, 2000)[14]. Once PSII function is impaired, photosynthetic electron transport and carbon assimilation decline, reducing the production of assimilates and metabolic energy. Heat-stressed seedlings may therefore enter an energy- and carbon-limited physiological state. This limitation may directly affect subsequent regeneration. During early organogenesis, excised stem segments require sufficient metabolic activity to support cell division, reprogramming, bud initiation, and shoot development. Although culture media provide external carbohydrates, regeneration still depends strongly on the physiological condition of donor tissues. Explants derived from seedlings with severe photosynthetic impairment may have lower metabolic vigor, altered redox balance, and reduced endogenous reserves, thereby weakening organogenic competence. Thus, a coherent pathway can be proposed: high temperature → PSII dysfunction → reduced carbon and energy acquisition → weakened physiological vigor → impaired regeneration.This relationship is particularly relevant to P. chinensis, which is sensitive to heat during acclimatization[9,10,12]. Similar temperature sensitivity has also been reported in Dendrobium regeneration (Teixeira da Silva et al., 2015)[15]. Therefore, testing whether post-stress Fv/Fm can predict regeneration capacity may provide both mechanistic insight and a practical tool for screening heat-tolerant germplasm.

In this study, six-month-old P. chinensis seedlings were used as material and subjected to four high-temperature stress treatments at 34℃, 36℃, 38℃, and 40℃[16]. During the 0-96h treatment period, we systematically measured the UV-Vis absorption spectra of stem segment extracts under different temperature treatments, quantitatively analyzed the dynamic changes in chlorophyll a, chlorophyll b, and total chlorophyll contents, and simultaneously monitored changes in Fv/Fm, the maximum photochemical efficiency of photosystem II[17]. Subsequently, stem segments subjected to different high-temperature treatments were inoculated onto induction media to evaluate their sprouting and regeneration capabilities, followed by correlation analyses between these physiological indicators and propagation potential[18]. This study aimed to characterize the damage response patterns of the photosynthetic system in P. chinensis tissue-cultured seedlings under high-temperature stress, reveal the relationship between photosynthetic physiological damage and regeneration capacity, and evaluate the feasibility of using spectral and chlorophyll fluorescence indicators for early stress diagnosis and heat-tolerant material screening. The findings are expected to provide a theoretical basis and technical reference for high-temperature-resistant tissue culture, rapid propagation, and cultivation management of P. chinensis and other orchids[19].

2. Materials and Methods

2.1. Plant Material and Culture Conditions

The experimental materials used in this study were provided by the Key Laboratory of Orchids, Fujian Agriculture and Forestry University. Six-month-old P. chinensis seedlings with similar age, height, growth vigor, and leaf color were selected as experimental materials and maintained in sterile culture bottles. The culture conditions were as follows: temperature of 25 ± 2℃, light intensity of 1500–2000 lx, photoperiod of 16 h/d, and relative humidity of 70% RH. The basal culture medium consisted of 1/2 MS (2.47 g/L) supplemented with sucrose (30 g/L), agar (7 g/L), activated charcoal (1 g/L), 6-BA (1.0 mg/L), NAA (0.2 mg/L), and potato juice (100 ml/L), with pH adjusted to 5.8–6.0.

2.2. High-Temperature Stress Treatment

P. chinensis seedlings with consistent growth status were individually transferred into sterile culture bottles containing medium and placed in a light incubator for high-temperature stress treatment. Four temperature treatments were applied: 34℃, 36℃, 38℃, and 40℃. All treatments were conducted with a photoperiod of 16 h/d, light intensity of 2000 lx, and relative humidity of 70% RH. The high-temperature stress treatment lasted for up to 96 h, and samples were collected at 0, 24, 48, 72, and 96 h for subsequent analyses.

2.3. UV-Vis Absorption Spectroscopy

During the high-temperature stress treatment, samples were collected from each treatment group every 24 h. Four seedlings subjected to high-temperature treatment were sampled. After removing the leaves and roots, the stem segments were retained as experimental materials, with approximately 0.5 g fresh weight used for each sample. Samples for subsequent analyses were rapidly frozen in liquid nitrogen and stored in the dark at −80℃ until use. Before measurement, the stem segments were placed in a glass Petri dish on ice and rapidly minced using a new, sharp razor blade. All minced stem segments were transferred into a 50 ml stoppered centrifuge tube, followed by the addition of 10 mL of 95% ethanol for extraction. The tube was tightly sealed and left at room temperature for 2.5 hours before filtration. The extract was then filtered and adjusted to a final volume of 10 mL when necessary. The extract was transferred into a 1 cm quartz cuvette, and 95% ethanol was used as the blank control. Scanning was performed on a UV-3000 UV–Vis spectrophotometer. The wavelength range was 350–700 nm, with a slit width of 1 nm and a scanning speed of 0.5 s. Absorbance values of stem segment extracts from P. chinensis under different durations of high-temperature stress were recorded across the 350–700 nm wavelength range. All measurements were repeated five times.

2.4. Chlorophyll Fluorescence Parameter (Fv/Fm)

During the high-temperature stress treatment, five seedlings were randomly sampled from each treatment group every 24 h. Fv/Fm was measured immediately using fresh living materials. Before measurement, high-temperature-treated seedlings were dark-adapted for 20 minutes. Chlorophyll fluorescence parameters of the stem segments were measured using a portable modulated chlorophyll fluorometer (PAM). The Fv/Fm value (Maximal quantum yield of PSII photochemistry) was recorded. All parameter measurements were repeated five times.

2.5. Assessment of Propagation Potential

To evaluate the effects of high-temperature stress on the propagation potential of P. chinensis seedlings, stem segments were collected immediately after each stress treatment and inoculated onto fresh induction medium. The induction medium had the same composition as the basal medium described in the “Plant material and culture conditions” section. After inoculation, the plants were placed in a sterile culture room under the following conditions: temperature 25 ± 2℃, light intensity 1500–2000 lx, and photoperiod 12 h/d. Each treatment included three biological replicates, with 10 stem segments per replicate. After 8 weeks of recovery culture, sprouting and regeneration of the stem segments were recorded, and the proliferation coefficient was calculated to evaluate the tissue culture regeneration ability of the materials after different high-temperature stress treatments.

2.6. Statistical Analysis

All data were statistically analyzed using SPSS 19.0 software. Two-way analysis of variance (ANOVA) was performed to examine the effects of temperature, stress duration, and their interaction on physiological indicators and propagation-related parameters. Correlation analysis was used to explore relationships between physiological indicators (e.g.; absorbance at characteristic wavelengths, Fv/Fm) and propagation potential parameters. A value of p < 0.05 was considered statistically significant. All results are presented as the mean ± standard deviation (mean ± SD). Physiological indicators were measured with five replicates, whereas the propagation potential assessment included three biological replicates.

3. Results

3.1. High-Temperature Effects on Absorbance of P. chinensis Seeding

High-temperature stress induced distinct alterations in the absorption spectra of pigment extracts from P. chinensis seedling stem segments (Figure 1). At 34℃, the extracts retained typical chlorophyll a (430 nm and 662 nm), chlorophyll b (453 nm and 643 nm), and carotenoid (400–500 nm) absorption features throughout the stress period, although peak intensities showed moderate reductions with prolonged treatment. In contrast, at higher temperatures (36℃, 38℃, and 40℃), the spectral profiles at 96 h did not exhibit uniform or sustained attenuation across all major absorption peaks. Notably, in the 430–460 nm and 650–670 nm regions, the 96-h absorbance values under 36℃ and 40℃ treatments were comparable to or even higher than those at early time points. This pattern—greater apparent pigment retention at higher temperatures than at 34℃—indicates that the relationship between temperature and pigment degradation is not simply monotonic, and suggests that distinct physiological mechanisms may operate under moderate versus acute heat stress[20].

Correlation analysis (Table 1) further revealed temperature- and time-dependent patterns in the coordinated behavior of pigment absorbance. At 34℃, absorbance values across the 350–700 nm range were predominantly significantly positively correlated (r > 0.60–0.90, P < 0.01) at most time points, suggesting a relatively synchronized degradation process. At 36℃ and above, correlation coefficients generally increased, particularly in the 400–500 nm and 600–700 nm regions, with many values exceeding r = 0.90 (P < 0.01). This strengthening of correlations at higher temperatures implies a more tightly coupled systemic response of the photosynthetic pigment system under severe stress, rather than independent or stochastic breakdown of individual pigment components. Regarding pigment-specific sensitivity, chlorophyll b-associated wavelengths (453 nm, 643 nm) remained highly positively correlated with chlorophyll a-associated wavelengths (430 nm, 662 nm) across all high-temperature treatments (r > 0.90, P < 0.01 in most cases), indicating coordinated but differentially rate-limited degradation between the two chlorophyll types. Carotenoid-region absorbance (400–500 nm) also showed strong synchronicity with chlorophyll peaks (r > 0.85, P < 0.01), further supporting the notion of concerted pigment system responses.

Interestingly, at 34℃, some wavelengths exhibited a slight increase in absorbance during the early stage (24 h), which may reflect a transient stress-responsive adjustment[21]. However, no consistent pattern of rapid and sustained attenuation from the onset was observed across all higher-temperature treatments; instead, the spectral changes were highly wavelength-specific and time-dependent. Notably, the gradual elevation of the spectral baseline, particularly in the 350–400 nm and 500–700 nm regions, was observed under certain higher-temperature treatments—most prominently at 40℃. This baseline shift is likely attributable to the accumulation of pigment degradation byproducts and membrane lipid peroxidation products[22], indicating that although bulk pigment content remained quantitatively high at elevated temperatures (as shown below in Figure 2), progressive structural and metabolic disruption of the photosynthetic apparatus was nevertheless occurring. This dissociation between spectral integrity (preserved peaks) and structural disruption (elevated baseline) foreshadows a key finding of this study: pigment content and photosynthetic functionality respond to heat stress through distinct mechanisms, a point that will be further substantiated by the Fv/Fm and proliferation data presented in subsequent sections.

3.2. Changes in Chlorophyll a, Chlorophyll b, and Total Chlorophyll Content Under High Temperature Stress

The quantitative analysis of chlorophyll content (Figure 2) confirmed the non-monotonic pattern suggested by the spectral data described above. Using the Arnon method[23] (Chl a = 13.95 × A₆₆₅ – 6.88 × A₆₄₉; Chl b = 24.96 × A₆₄₉ – 7.32 × A₆₆₅; Total Chl = Chl a + Chl b, where A₆₄₉ and A₆₆₅ are absorbance values at the corresponding wavelengths), we quantitatively analyzed the dynamic changes in chlorophyll a, chlorophyll b, and total chlorophyll content in stem segments under high-temperature stress.The results showed that the effect of high-temperature stress on the chlorophyll system exhibited a distinct and unexpected temperature-dependent pattern (Figure 2). Under 34℃ treatment, chlorophyll content showed a progressive and substantial decline with prolonged stress duration, with total chlorophyll decreasing to approximately 40% of the initial value at 96 h—representing the most substantial reduction among all treatments[24]. In striking contrast, the 36℃, 38℃, and 40℃ treatment groups exhibited relatively minor fluctuations in chlorophyll content over the 96-h period, generally maintaining 70%–95% of their initial values, without showing sustained or drastic degradation. Notably, the 40℃ treatment group showed only slight fluctuations in chlorophyll content within the first 24 h and still retained more than 85% of the initial value at 96 h, forming a marked contrast with the 34℃ treatment group. Further analysis of the Chl a/b ratio revealed inconsistent trends among treatment groups, with no monotonic increase observed as temperature rose[25], suggesting that the differential sensitivity of chlorophyll a and b to heat stress may be modulated by factors beyond temperature alone. Collectively, these quantitative data confirm that not all high-temperature treatments lead to a linear or predictable collapse of the chlorophyll system. Instead, 34℃—while only moderately elevated relative to the optimal culture temperature (25℃)—unexpectedly triggered more pronounced chlorophyll degradation than did higher temperatures (36–40℃).

The relative stability of chlorophyll content under acute higher-temperature stress (36–40℃) suggests that P. chinensis seedlings may activate rapid protective responses—such as heat shock protein induction or antioxidant systems—that transiently preserve pigment integrity under short-term extreme heat. However, as will be demonstrated by the Fv/Fm data in the following section, this quantitative stability of pigment content does not equate to functional integrity of the photosynthetic apparatus. The apparent preservation of chlorophyll at 40℃ masks severe impairment of PSII photochemical efficiency, underscoring the critical distinction between structural retention of pigment molecules and the maintenance of photosynthetic function. This dissociation represents a key finding of the present study and will be explored further in the context of regenerative capacity[26].

Figure 2. Changes in chlorophyll a/b ratio and total chlorophyll content in P. chinensis seedling stem segments under high-temperature stress.

Figure 2. Changes in chlorophyll a/b ratio and total chlorophyll content in P. chinensis seedling stem segments under high-temperature stress.

3.3. Changes in Fv/Fm Values of Stem Segments of P. chinensis Seedlings Under High-Temperature Stress

While the preceding section revealed that chlorophyll content remained quantitatively stable at higher temperatures (36–40℃), this apparent stability did not reflect the true functional state of the photosynthetic apparatus. The maximum photochemical efficiency of PSII (Fv/Fm)—a key indicator of the primary light energy conversion capacity and the intrinsic activity of PSII reaction centers[27]—was therefore measured to assess functional impairment under heat stress. Under non-stress conditions, the baseline Fv/Fm value of P. chinensis seedlings was 0.74 ± 0.023, slightly below the theoretical optimal range of 0.80–0.85 typically observed in fully acclimated mature plants[28]. This modestly lower baseline may be attributed to their in vitro culture state, incomplete acclimation, or the measurement site (stem segments rather than mature leaves)[29,30], but served as a consistent reference for comparing stress-induced changes.

High-temperature stress significantly inhibited Fv/Fm in P. chinensis seedlings, and the degree of inhibition was positively correlated with both stress temperature and treatment duration (Table 2; Figure 3). Under 34℃ treatment, Fv/Fm slightly increased to 0.77 at 24 h, exhibiting a transient "hormesis effect," followed by a continuous decline to 0.55 at 48 h; it remained between 0.50 and 0.59 from 72 to 96 h, indicating structural damage to PSII with limited recovery capacity. Under 36℃ treatment, Fv/Fm decreased progressively to 0.40 at 72 h, then showed a modest recovery to 0.45 at 96 h, suggesting that the PSII repair mechanism, although severely challenged, remained partially functional[31]. In contrast, under 38℃ treatment, Fv/Fm declined steadily throughout the stress period, reaching 0.41 at 96 h with no clear recovery trend, indicating that the critical threshold for irrecoverable damage lies between 36℃ and 38℃, with 38℃ representing the temperature beyond which protective mechanisms fail and sustained suppression ensues. The most severe impairment occurred at 40℃: Fv/Fm dropped to 0.52 at 24 h and further to only 0.25 at 96 h—far below the commonly recognized photoinhibition threshold (approximately 0.70)—indicating large-scale inactivation or irreversible destruction of PSII reaction centers.

The differential Fv/Fm responses across temperatures observed in this study—partial recovery at 36℃ versus sustained suppression at 38℃ and catastrophic decline at 40℃—are consistent with multi-layered damage to the PSII complex induced by heat stress. Such damage includes disruption of D1 protein conformation and function, uncoupling of thylakoid membrane structure leading to impaired energy transfer[32], and inactivation of heat-sensitive manganese clusters in the oxygen-evolving complex affecting electron supply[33]. These lesions accumulate with increasing temperature and prolonged stress duration, ultimately triggering irreversible decline in PSII function when repair mechanisms are overwhelmed[34,35]. Notably, the contrast between the Fv/Fm data presented here and the pigment content data shown in the previous section is striking: while chlorophyll content remained quantitatively stable at 40℃, Fv/Fm collapsed to 0.25, demonstrating that pigment retention and photosynthetic functionality are dissociated under acute severe heat stress. This dissociation has direct implications for regenerative capacity, as explored in the following section. In summary, Fv/Fm served as a rapid, sensitive, and physiologically relevant indicator for assessing the degree of heat stress and the photosynthetic functional state of P. chinensis seedlings. The sustained decline in PSII photochemical efficiency under high-temperature stress directly reveals the vulnerability of its photosynthetic apparatus to thermal stress[36]. This functional impairment—rather than pigment loss per se—represents the primary physiological bottleneck restricting stress tolerance and regenerative potential[37,38].

3.4. Correlation Between Fv/Fm Values and Tissue Culture Potential of Stem Segments in P. chinensis Seedlings Under High-Temperature Stress

The preceding section established that Fv/Fm reveals functional impairment that is not reflected by pigment content alone. To determine whether this functional impairment translates into reduced regenerative capacity, stem segments from P. chinensis seedlings subjected to different temperature treatments were inoculated onto proliferation medium. After 8 weeks of recovery culture, the proliferation coefficient was recorded and subjected to correlation analysis with the Fv/Fm values measured during the stress period (Table 3). Morphological observations were consistent with the physiological data. Seedlings under 34℃ and 36℃ treatments retained relatively green coloration and structural integrity throughout the 96-h stress period, although slight leaf wilting was observed at 36℃. In contrast, 38℃ treatment induced progressive leaf chlorosis and wilting, while 40℃ treatment resulted in severe necrosis and tissue browning as early as 72 h, with complete collapse by 96 h (Figure 4).

The results showed a highly significant positive correlation between Fv/Fm values and stem segment proliferation capacity (r > 0.85, P < 0.01), confirming that Fv/Fm can serve as an effective indicator for predicting regenerative potential[39,40]. A more detailed examination of the temperature-specific responses revealed the following patterns (Table 3). Under 34℃ treatment, Fv/Fm ranged from 0.50 to 0.59, with proliferation coefficients between 2.32 and 3.43—representing the highest regenerative capacity among all stressed treatments. Under 36℃ treatment (Fv/Fm = 0.40–0.45), proliferation coefficients ranged from 2.20 to 3.22, indicating that despite severe transient PSII suppression at 72 h (Fv/Fm = 0.40), the partial recovery observed at 96 h (Fv/Fm = 0.45) was accompanied by measurable regenerative capacity. Under 38℃ treatment (Fv/Fm = 0.41–0.58), proliferation coefficients declined to 1.70–3.37, with the lowest value (1.70 at 96 h) corresponding to sustained Fv/Fm suppression without recovery. Under extreme high-temperature stress at 40℃, Fv/Fm dropped to 0.25 at 96 h, and proliferation capacity was completely lost (proliferation coefficient = 0), indicating that a threshold of irreversible damage had been exceeded. Notably, all 24-h treatments—regardless of temperature—retained relatively high proliferation coefficients (2.40–3.37), suggesting that short-term heat exposure (≤24 h) caused limited lasting damage to regenerative capacity, whereas prolonged stress (≥48 h) progressively eroded regeneration potential in a temperature-dependent manner[41].

Based on the observed distribution, a preliminary response threshold was estimated: when Fv/Fm exceeded approximately 0.55, proliferation coefficients generally remained above 2.0 (e.g.; most 34℃ treatments and 24-h treatments across all temperatures); when Fv/Fm ranged between 0.40 and 0.55, proliferation was reduced but still detectable (coefficients 1.7–2.2); and when Fv/Fm fell below 0.40, proliferation was severely compromised (coefficient < 1.0) or completely lost (coefficient = 0 at Fv/Fm = 0.25). A notable exception was observed at 34℃-96h (Fv/Fm = 0.50, proliferation coefficient = 2.32), suggesting that under moderate prolonged stress, regenerative capacity may partially recover through mechanisms not directly captured by Fv/Fm alone, such as metabolic repair during subsequent culture.

The strong association between Fv/Fm and regenerative capacity observed in this study is consistent with multi-level physiological interdependencies. Fv/Fm directly reflects the functional state of PSII and the integrity of the photosynthetic electron transport chain, which governs the supply of ATP and NADPH[42,43]—both of which are critically required for the energy-intensive processes of cell division and organogenesis during regeneration. Additionally, a decline in Fv/Fm is often accompanied by photoinhibition and reactive oxygen species (ROS) burst, which can cause oxidative damage to cellular membranes and DNA, thereby inhibiting cell viability and totipotency expression[44,45]. Conversely, cells maintaining higher Fv/Fm values are likely to possess more robust heat protection mechanisms and greater recovery capacity[46,47]. Thus, Fv/Fm serves as a comprehensive physiological integrator that reflects not only photosynthetic function but also the overall metabolic and stress-resilience status of the explant. In summary, a strong positive correlation exists between Fv/Fm values and the tissue culture potential of stem segments from P. chinensis seedlings. By monitoring Fv/Fm, the regenerative potential of explants can be predicted non-destructively and rapidly before inoculation, providing a reliable technical means for early screening and quality assessment of tissue culture materials[48]. This finding reveals—from a photosynthetic physiological perspective—the mechanistic link between heat-induced PSII damage and reduced regenerative capacity, directly addressing a practical question in tissue culture: why do heat-stressed seedlings exhibit poor survival and regeneration. The application of Fv/Fm provides a theoretical basis and quantifiable operational indicators for optimizing the rapid propagation system of P. chinensis, particularly for developing science-based seedling production management strategies under high-temperature stress conditions[49,50].

4. Discussion

4.1. Heat-Induced Changes in Pigment Content, PSII Function, and Regenerative Capacity

This study examined the responses of in vitro P. chinensis seedlings to short-term high-temperature treatments by integrating absorbance-based pigment estimates, Fv/Fm measurements, and subsequent stem-segment proliferation. The principal finding was that these indicators did not respond identically to increasing temperature. Pigment-related indices showed non-monotonic variation across temperatures and treatment durations, whereas Fv/Fm showed increasingly severe suppression at the 96-h endpoint. In addition, lower Fv/Fm values were generally associated with lower subsequent proliferation coefficients[51,52]. We therefore propose that Fv/Fm can serve as an early functional indicator for predicting regeneration potential in heat-stressed P. chinensis seedlings, offering a practical framework for screening heat-tolerant germplasm during acclimatization. Nevertheless, the non-monotonic pigment response should be interpreted cautiously. Spectrophotometric chlorophyll estimates depend directly on absorbance at selected wavelengths and may be influenced by baseline shifts, tissue browning, light scattering, or the accumulation of non-chlorophyll compounds. Because a pronounced baseline elevation was observed under some severe heat treatments, particularly at 40℃, the apparently high chlorophyll estimates under these conditions cannot be considered conclusive evidence of pigment protection. The present data therefore demonstrate a difference between absorbance-based pigment estimates and PSII functional responses, but they do not establish the biochemical mechanism responsible for this difference. Fv/Fm provided clearer evidence of functional impairment under severe heat treatment. At the 96-h endpoint, Fv/Fm decreased progressively from 0.50 at 34℃ to 0.25 at 40℃. However, several intermediate time points showed non-monotonic responses, demonstrating that the effects of temperature depended on exposure duration. The modest increase observed between 72 and 96 h at 36℃ may indicate partial physiological adjustment during treatment, but it cannot be regarded as evidence of complete PSII repair because fluorescence was not measured after a subsequent recovery period. Similarly, the sustained decline observed at 38℃ indicates stronger suppression during the treatment period but does not, by itself, demonstrate irreversible damage. The positive relationship between Fv/Fm and the proliferation coefficient suggests that PSII status is associated with the subsequent regenerative performance of stem segments. However, this association should not be interpreted as direct evidence that reduced PSII efficiency caused the decline in regeneration. Fv/Fm may instead function as an integrated indicator of overall physiological vigor, membrane stability, oxidative status, and cellular damage (Baker and Rosenqvist, 2004)[53]. The wide range of proliferation coefficients observed at similar Fv/Fm values further indicates that regenerative capacity is controlled by multiple physiological processes and cannot be predicted from a single fluorescence value.

4.2. Dissociation Between Chlorophyll Content and Photosynthetic Function Under Heat Stress

This functional interpretation is further strengthened by one of the most striking findings of this study: the marked dissociation between pigment content and photosynthetic functionality. Quantitative pigment data revealed that total chlorophyll decreased most substantially at 34℃, falling to approximately 40% of the initial value at 96 h, whereas at higher temperatures (36–40℃) it remained relatively stable, retaining 70%–95% of initial values throughout the stress period. In contrast, Fv/Fm declined in a temperature-dependent manner, with the most severe impairment at 40℃ (Fv/Fm = 0.25 at 96 h) and a comparatively milder reduction at 34℃ (Fv/Fm = 0.50). Thus, the temperature that caused the least apparent pigment loss (40℃) produced the most severe functional damage, whereas the temperature associated with the greatest pigment loss (34℃) resulted in relatively better-preserved PSII function and higher regenerative capacity, with proliferation coefficients of 2.32–3.43 compared with complete loss of proliferation at 40℃. Further analysis of the Chl a/b ratio revealed inconsistent trends among treatment groups and no monotonic increase with increasing temperature[54,55], suggesting that the differential sensitivity of chlorophyll a and b to heat stress may be modulated by factors beyond temperature alone. This dissociation can be explained by considering the distinct temporal dynamics and mechanistic targets of heat damage. Pigment degradation primarily reflects cumulative oxidative damage to chlorophyll molecules and their associated light-harvesting complexes[56,57], a process that may be accelerated under prolonged moderate stress (34℃) through senescence-associated pathways (Hörtensteiner and Kräutler, 2011)[58]. By contrast, PSII functional decline, as reflected by Fv/Fm, arises from more immediate and structurally specific lesions, including D1 protein denaturation, disruption of the oxygen-evolving complex, and uncoupling of thylakoid membrane organization [59,60], which can occur even when bulk pigment content remains quantitatively intact (Allakhverdiev et al., 2008)[61]. PSII reaction centers are embedded in the thylakoid membrane, and their damage involves physical conformational changes that may outpace the capacity of protein repair machinery. At 40℃, rapid induction of heat shock proteins and enzymatic antioxidant systems may transiently protect pigment molecules from oxidative bleaching, but these protective mechanisms appear insufficient to preserve the structural integrity of PSII reaction centers, which are inherently more thermolabile. This interpretation is also consistent with the spectral baseline elevation observed at 40℃, which may indicate the accumulation of degradation byproducts and membrane lipid peroxidation products despite the maintenance of apparently intact pigment content. The strong positive correlation between Fv/Fm and subsequent proliferation coefficient (r > 0.85, P < 0.01) observed in this study is consistent with findings in other plant systems, where photosynthetic efficiency serves as a reliable proxy for overall physiological vigor and regenerative potential[56]. Our study extends this relationship to a medicinal orchid and further indicates that Fv/Fm[62], rather than pigment content, is the physiologically relevant predictor of regenerative capacity (Kalaji et al., 2016)[63]. This is evidenced by the fact that 34℃-treated seedlings, despite suffering the most severe pigment loss, retained relatively high proliferation coefficients, whereas 40℃-treated seedlings, despite maintaining pigment content above 85%, exhibited complete loss of regenerative capacity at 96 h. Collectively, these results indicate that PSII functional integrity is more closely associated with subsequent organogenic capacity than bulk pigment content in this species.

4.3. Differential Physiological Responses at 36℃ and 38℃ Reveal a Potential Thermal Threshold

Another notable finding was the differential response between the 36℃ and 38℃ treatments. At 36℃, Fv/Fm decreased progressively to 0.40 at 72 h and then showed a modest recovery to 0.45 at 96 h, accompanied by measurable regenerative capacity, with proliferation coefficients ranging from 2.20 to 3.22. In contrast, at 38℃, Fv/Fm declined steadily throughout the stress period without a clear recovery trend, and proliferation was more severely compromised, with coefficients ranging from 1.70 to 3.37 and the lowest value occurring at 96 h. These results suggest that a critical transition zone for recoverable versus severe heat damage may lie between 36℃ and 38℃ in P. chinensis seedlings. The partial recovery observed at 36℃ may reflect the activation of protective mechanisms such as heat shock proteins, membrane lipid remodeling[64], and enhanced chlorophyll biosynthesis that remain effective within this temperature range, whereas at 38℃ these mechanisms may become insufficient to counteract accumulated damage, resulting in sustained physiological suppression without clear recovery. This pattern is reminiscent of hormesis-like responses observed in other plant species under temperature stress (Agathokleous et al., 2019)[65]. Compared with heat-tolerant crops such as rice, which can maintain a certain degree of photosynthetic activity at approximately 38℃ through heat-protective mechanisms, P. chinensis exhibited substantially greater physiological impairment at the same temperature, suggesting that its metabolic tipping point may be lower than that of many crop species. This may indicate a distinct high-temperature adaptation strategy that warrants further molecular investigation. Such a narrow thermal safety margin also highlights the vulnerability of this epiphytic orchid to moderate warming events, which could directly threaten propagation success in conservation and nursery programs.

4.4. Fv/Fm as a Practical Indicator for Heat-Stress Assessment and Seedling Management

Our findings further suggest that the boundary between acclimatory and damaging stress is not fixed but dynamically shifts depending on stress duration and the temporal dynamics of physiological responses (Mittler, 2006; Zandalinas et al., 2018)[66,67,68,69]. The identification of a discrete critical temperature zone between 36℃ and 38℃, rather than a simple continuous gradient effect[70], provides empirical support for threshold-based models while also revealing that pigment-based and function-based indicators may yield fundamentally different threshold estimates. Pigment content suggested relative stability across 36–40℃, whereas Fv/Fm revealed a much clearer functional transition between 36℃ and 38℃. This distinction has direct practical significance because relying on pigment content as the sole indicator of heat tolerance could be misleading, particularly under acute high-temperature stress, where apparent pigment stability may mask substantial PSII impairment. Our results therefore suggest that chlorophyll fluorescence, particularly Fv/Fm, can serve as a rapid and non-destructive diagnostic tool for the early detection of heat stress and for estimating regenerative potential before inoculation. Controlled and phased high-temperature hardening [71], for example, exposure to 36℃ for up to 96 h, could potentially be developed as a technique to enhance seedling robustness[72], provided that appropriate recovery periods are incorporated. In contrast, prolonged exposure to temperatures at or above 38℃should be avoided because it is associated with sustained PSII suppression and a pronounced reduction in regenerative potential.

4.5. Future Research Directions and Implications for P. chinensis Conservation

Future studies should integrate transcriptomic and proteomic approaches[73] and leverage emerging biotechnological tools such as CRISPR-based gene editing [74] to identify the key genes and metabolic pathways that distinguish successful acclimation at 36℃ from physiological failure at 38℃[75]. Such studies should particularly focus on heat shock proteins, antioxidant systems, thylakoid membrane stability, PSII repair processes, and pathways involved in cellular regeneration. Furthermore, because natural stress conditions usually involve multiple interacting environmental factors, future studies should also investigate the combined effects of heat and drought, which would more realistically reflect field conditions[76]. Recovery experiments following heat exposure will also be necessary to distinguish transient PSII suppression from truly irreversible damage and to determine whether the apparent recovery observed at 36℃ translates into sustained physiological and regenerative advantages. In summary, this study provides a physiological basis for using chlorophyll fluorescence as a management and screening tool in orchid conservation and tissue-culture propagation and identifies a practical upper temperature range that should be considered during nursery production of P. chinensis.