Abstract
Denphal-type Dendrobium is the famous cut and potted flower in the world, and most cultivated in tropical and subtropical regions. However, it often suffers from cold in winter in subtropical regions. To verify the physiological response of Denphal-type Dendrobium under low temperature in this study, the mature and young plantlets of Dendrobium Udomsri Beauty were treated under 15, 10, and 5 °C, respectively. And then the electrical conductivity (EC), soluble protein, soluble sugar, free proline, malondialdehyde (MDA), chlorophyll content, and the rate of defoliation after regrowth were measured. The results showed that both mature plant and young seedlings of Dendrobium Udomsri Beauty, the EC, soluble protein, soluble sugar, free proline, MDA content, and defoliation rate were increased with the decrease of treatment temperature and the extension of treatment times. The content of chlorophyll decreased gradually with the decrease of treatment temperature and the extension of treatment times. The correlation analysis showed that soluble sugar, free proline, MDA, chlorophyll content, and defoliation rate were significantly correlated with the semi-lethal temperature. It is indicated that the content of free proline, MDA, chlorophyll, and defoliation rate could be used as the effective indexes for the comprehensive assessment of cold tolerance of Dendrobium Udomsri Beauty.
Low temperature is a pivotal environmental factor influencing plant growth, development, and distribution (Boyer 1982). Low temperature is a substantial abiotic stressor impacting plants in diverse ways, leading to stunted plant structure and development, disrupted physiological metabolism, compromised photosynthetic efficiency, and damage to cell membranes that result in the leakage of ions and soluble substances (Banerjee et al. 2017). To counter low-temperature stress, plants employ various strategies, including modifications to the cell membrane system, enhancement of antioxidant mechanisms, osmotic regulation, metabolic adaptations, and hormone regulation (Xie et al. 2020).
Low temperatures directly inhibit plant metabolic reactions and indirectly induce membrane permeability changes, affecting water absorption and leading to cell dehydration. Low temperatures can also cause physiological damage to roots, reducing the water supply to leaves. This decrease in leaf water content can lead to cellular water deficiency, resulting in necrotic spots, leaf wilting, stunted growth, and impeded sprouting, among other issues (Gai 2008).
The Dendrobium genus is the second largest genus in the Orchidaceae and is highly significant due to its commercial cultivation of tropical orchids, which is primarily used for cutting flowers and pot flowers (Ketsa and Warrington 2023). During winter, there is a substantial surge in demand for cut orchid flowers in Europe, United States, Japan, and other primary importing countries in the northern hemisphere, coinciding with reduced local production (Ketsa and Warrington 2023). Simultaneously, low temperatures during this season limit the flowering of orchid plants cultivated commercially across several Asian countries.
Research has shown that Dendrobium inflorescences appear to be more sensitive to low temperature than either Phalaenopsis or Cymbidium (American Orchid Society 1985; Huang et al. 1988). Exposing them to 10 °C for more than 4 d or 7.5 °C for more than 2 d triggers chilling injury (CI) in Dendrobium (Dai and Paull 1991).
The impact of low-temperature stress on flower growth and development varies significantly depending on factors such as plant variety, geographical location, stress intensity, and the method of stress application. Plants exhibit diverse physiological mechanisms and resistance strategies to cope with low-temperature stress, including changes in photosynthetic activity, osmotic regulation, scavenging of reactive oxygen species, and structural modifications in leaves. Despite substantial advancements in understanding plant responses to low temperatures, further investigation is needed to explore the comprehensive effects of combined environmental factors and their interactions with other variables (Xie et al. 2020).
Ambient temperatures play a crucial role in both the flowering and vegetative growth phases of Dendrobium spp. plants. Optimal growth for Dendrobium spp. and their hybrids occurs when night temperatures stay above 18 °C, with daytime temperatures ranging between 24 ° and 29 °C. Typically, colder night temperatures (below 15 °C) impose more significant limitations on orchid plant growth and development compared with daytime temperatures. Decreased daytime temperatures result in leaf discoloration, foliage loss, and diminished vegetative growth. Moreover, lower temperatures and shorter daylight periods have been observed to alter the concentration of internal growth regulators, prompting the initiation of flowering in sympodial orchids (De et al. 2013). The temperature needs for flowering potted plants align closely with those producing cut flowers. Therefore, it is crucial to investigate the cold tolerance of garden plants and explore methods to enhance their resilience to low temperatures.
This study assessed the cold tolerance of Dendrobium Udomsri Beauty seedlings under low temperatures. Correlation analysis identified soluble sugars, free proline, MDA, chlorophyll, and leaf fall rate as comprehensive indicators for cold resistance. This research lays groundwork for assessing and managing cold resilience in Dendrobium production. In addition, it advances the understanding of Dendrobium's adaptation to cold climates, aiding in improved cultivation strategies.
Materials and Methods
Plant material
The Dendrobium Udomsri Beauty, available as both mature plants and young seedlings (3 inches, 10 × 11 × 8.5 cm), were cultivated in coconut husk growing medium at the Tropical Orchid Resource Nursery, part of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science. These 6-month-old seedlings with height ranging from 20 to 25 cm and six to eight leaves, were put into distinct artificial climate chambers. These chambers were set to maintain temperatures at 5, 10, and 15 °C for the low-temperature treatment, respectively, alongside a 14-h light and 10-h dark photoperiod at an illumination intensity of 20,000 lx, the relative humidity remained at 85%.
Physiological measurements
Determination of relative conductivity.
The Dendrobium plants were situated within an artificial climate chamber, in which a temperature gradient of 10, 5, 0, −5 °C, temperature treatment 0 (CK), 1 h, 2 h, 4 h, 8 h, 24 h, and 2 d, 4 d, 8 d, and 16 d was established for a 12-h low-temperature treatment. Light exposure was regulated at a 14-h light to 10-h dark ratio, with a light intensity set at 20,000 lx and air humidity maintained at 85%. For comparison, three fresh, fully expanded leaves from the top were randomly selected for each treatment. The conductivity measurements were repeated three times for each treatment.
Determination of physiological indicators.
The measurement of chlorophyll content was carried out by a spectrometric method. The MDA content was measured using the thiobarbituric acid method (Heath and Packer 1968). Free proline (FPro) content was measured using the acidic ninhydrin method (Bates et al. 1973). Soluble sugars were quantified using the anthrone method (Yemm and Willis 1954), and soluble protein was measured through the Coomassie Brilliant Blue Method (Bradford 1976). Relative conductivity was gauged using the immersion method (Arnon 1949).
Statistical analysis
The analysis of variance was conducted using SPSS Statistics 26 (IBM, Armonk, NY, USA). Graphs and tables were generated using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Each measurement represents five biological replicates and three technical replicates. Duncan’s multiple range tests were used to determine significant differences between means, with P < 0.05 as the criterion for statistical significance.
Results
Statistical analysis
Effect of different low-temperature stress on soluble protein content.
Soluble protein serves as a crucial osmotic regulator in plants under low-temperature stress, with its content showing a positive linear correlation with cold tolerance and increasing under stress conditions (Fig. 1A and B). Following exposure to various temperature regimens, there was an initial modest surge, peaking at 2 to 4 h, followed by a gradual decline until 1 d. Subsequently, soluble protein levels gradually increased, reaching a peak at 2 d, then declined until 8 d. Another increment was observed, with the maximum value recorded at 16 d. In medium-sized seedlings of Dendrobium Udomsri Beauty treated at 15 °C for 16 d, the soluble protein content reached 402.78 µg/g.
(A, C, E, G, and I) Measurement of the soluble protein, soluble sugar, malondialdehyde, chlorophyll, and free proline content for mature plant. (B, D, F, H, and J) Measurement of the soluble protein, soluble sugar, malondialdehyde, chlorophyll, and free proline content for young seeding.
Citation: HortScience 59, 9; 10.21273/HORTSCI17974-24
Effects of different low-temperature stress on soluble sugar content.
During the treatment period at various temperature levels, the soluble sugar content showed a consistent increase with prolonged treatment duration. A substantial surge was observed after the fourth day of treatment, reaching its peak on the 16th day of exposure. Notably, the soluble sugar content in mature plants reached 1.1974 mg/g after 16 d of treatment at 15 °C (Fig. 1C and D).
Effect of low-temperature stress on MDA content.
The MDA content exhibited noticeable fluctuations, demonstrating a consistent upward trend with prolonged stress duration and escalating stress temperatures. Under various temperature treatments, the MDA content of Dendrobium Udomsri Beauty exhibited regular fluctuations, showing an upward trajectory with extended treatment duration and culminating at its peak on the 16th day. Particularly noteworthy in the Dendrobium Udomsri Beauty variant was the sharp increase in MDA content after 2 d, reaching a range of 13 to 28 nmol/g following the 16-d treatment (Fig. 1E and F).
Effects of different low-temperature stress on chlorophyll content.
Upon exposure to varying temperatures, the chlorophyll content showed consistent decline with the progression of the treatment duration. After being treated 16 d, all samples exhibited their lowest chlorophyll content. Across the same treatment temperatures, chlorophyll content showed a notable decline after 4 d treatment, particularly evident at treatment temperatures of 10 and 5 °C (Fig. 1G and H).
Effect of low-temperature stress on free proline content.
The free proline content initially showed a gradual decrease, followed by a notable increase with prolonged stress time and treatment temperature decreases. Specifically, there was a slower change in free proline content observed from 1 h to 8 d, with a sharp increase noted between 8 and 16 d. The proline content ranged from 4.84 to 14.03 μg/g. Notably, the free proline content showed the most significant differences in both young seedling and mature plant at 5 °C (Fig. 1I and J).
Effect of low-temperature stress on EC.
The relative conductivity of Dendrobium Udomsri Beauty showed a progressive elevation with the decreasing treatment temperatures, indicating an increasingly detrimental effect of low temperatures on cell membrane integrity. This gradual increase in membrane permeability resulted in a continuous elevation in EC, reaching its peak at −5 °C, marking the most severe damage induced by low temperature stress at this degree. Remarkably, throughout the low-temperature treatment process, the young seedlings consistently exhibited lower conductivity compared with the mature plants, suggesting a higher level of cold resistance in the Dendrobium Udomsri Beauty young seedlings (Fig. 2A and B).
The electrical conductivity of mature plants (A) and young seedlings (B) at different temperatures.
Citation: HortScience 59, 9; 10.21273/HORTSCI17974-24
Effect of low-temperature stress on injury index.
With the treatment temperature decreases and the treatment time prolongs, the yellow leaf rate and defoliation rate of Dendrobium Udomsri Beauty increase gradually. Under identical treatment temperatures, the yellow leaf rate of treated samples initially resembled that of nontreated samples within 8 h of treatment. However, after 1 d of treatment, the yellow leaf rate of treated samples gradually escalated, reaching its peak after 16 d (Figs. 3A–B and 4).
The yellow leaf rate of mature plants (A) and young seedlings (B) at different temperatures.
Citation: HortScience 59, 9; 10.21273/HORTSCI17974-24
Cold case of Dendrobium seedling cultivar.
Citation: HortScience 59, 9; 10.21273/HORTSCI17974-24
Correlation analysis of cold tolerance indexes of Dendrobium Udomsri Beauty.
There is a significant positive correlation between soluble sugar and MDA content, similar to the relationship observed between free proline and MDA content. Conversely, notable negative correlations were evident between soluble sugar content and defoliation rate, mirroring the association seen between free proline and defoliation rate. The negative correlation between defoliation rate and chlorophyll content stood out prominently. In addition, the interactions among chlorophyll content and soluble sugar, free proline, MDA content, and defoliation rate consistently displayed significantly or extremely negative correlations. Notably, no significant correlation was found between soluble protein and other cold-resistance indicators (Fig. 5).
The correlation between physiological and morphological indexes of Dendrobium ‘Udomsri Beauty’ mature (A) and young (B) plantlets under 5 °C different degrees of low-temperature stress. The different colors represent the value of the correlation, and the size of the circles in the figure indicates the absolute value of the correlation.
Citation: HortScience 59, 9; 10.21273/HORTSCI17974-24
Discussion
Following cold treatment, MDA levels exhibit fluctuation and a tendency to increase with prolonged stress duration, especially under lower temperatures. When subjected to stress, plant organs frequently experience membrane lipid peroxidation, with MDA as the end product of this degradation process. During exposure to low temperatures, there is a pronounced increase in MDA levels, which compromises the integrity of the cellular membranes. This escalation in MDA concentration correlates with more extensive membrane damage, thereby signaling reduced cold tolerance in the affected plant tissues (Kim et al. 2022; Liang et al. 2022; Qin et al. 2023; Tsikas 2017; Zhang et al. 2021). The increased synthesis of proline and MDA in stressed in plants under stress not only facilitates their recovery but also functions as a critical defense mechanism vital for survival (Ghars et al. 2008). This is consistent with the research findings in cotton (Dai et al. 2024).
Soluble sugars, crucial components of plant osmotic pressure, are highly responsive to environmental stresses. This study confirms that in Phalaenopsis-type Dendrobium, across different seedling stages, soluble sugar levels increase following exposure to cold stress. This response parallels observations in various ornamental plants such as waterlily, kale, and Rosa chinensis, all of which exhibit increased osmotic solutions under stress conditions, potentially enhancing plant resilience (Cheng et al. 2022; Jurkow et al. 2019; Mou et al. 2021). Further insights from research into the relationship between cold resistance and carbohydrates across diverse plant species highlight the protective roles of fructose and sucrose. These sugars are particularly effective in promoting winterhardiness, as demonstrated in plants like alfalfa, thereby underscoring their critical function in stress adaptation mechanisms (Sun et al. 2021). Furthermore, glucose and fructose levels have been identified as promising indicators of cold tolerance in Mediterranean pine. In a related study, when Xanthoceras sorbifolia seedlings were exposed to temperatures of 4 °C, a sustained elevation in both soluble sugar and soluble protein levels was observed over a 48-h period. This finding underscores the critical role of these biomolecules in enhancing seedling resilience against environmental adversities (Yan et al. 2020).
This study determined the soluble protein content in the leaves of Dendrobium under different temperatures and durations of low-temperature stress. The results showed that the soluble protein content exhibited regular fluctuations in response to prolonged stress periods. This pattern was consistent with the variation in soluble protein content observed in rhododendrons under natural low-temperature conditions (Wang et al. 2023). The observed fluctuations in soluble protein content in response to low-temperature stress may be attributed to an initial triggering of plant stress response mechanisms, resulting in an increase in soluble protein levels. As plants acclimate to prolonged stress, the soluble protein content subsequently decreases. However, continuous long-term exposure to low temperatures induces a cyclic response in plants, transitioning between stress response and adaptation phases. This cyclic pattern leads to systematic changes in soluble protein content characterized by multiple fluctuations. Interestingly, the variation pattern of soluble protein content in Phalaenopsis-type Dendrobium showed a positive correlation with LT50, although this correlation was not statistically significant. Consequently, soluble protein content was not identified as a key determinant of cold tolerance in Phalaenopsis-type Dendrobium based on this study’s findings.
This study investigated the free proline content in Dendrobium leaves under various low-temperature stresses and durations. The results revealed a significant increase in free proline content with prolonged and intensified low-temperature stress. Initially, this change occurred gradually, but as stress levels intensified, the magnitude of the increase in free proline content became more pronounced, indicating a robust cold-resistance response in the plants. The response of proline levels to low-temperature stress exhibits variability across plant species. For instance, under similar conditions, plants like Poa pratensis, rice, and Photinia pratensis also demonstrate a notable increase in proline content, highlighting a conserved adaptive mechanism among diverse plant species to cope with low-temperature stress (Dionne et al. 2001). During the low-temperature treatment, both fruit cane and Petunia initially displayed an increase in proline content, which was followed by a subsequent decrease over time. Notably, despite this decline, proline levels remained elevated throughout the treatment (Chen et al. 2016; Ning et al. 2016). Cold-tolerant varieties exhibited significantly higher proline content compared with their cold-sensitive counterparts in response to low-temperature stress. This observation underscores the role of proline as a potential biochemical marker for cold tolerance in plants, highlighting its importance in enhancing resilience against adverse environmental conditions (Xu et al. 2016). Nawaz et al. (2019) proposed that the principal divergence between cold-tolerant and cold-sensitive Pinus medusa seedlings lay in their varying capacities to accumulate osmotic regulators, particularly proline. Remarkably, the knockout of OsPRP1, a gene responsible for encoding proline-rich proteins in rice, notably increased plant susceptibility to cold stress. Xie et al. (2009) conducted a study involving 12 pepper cultivars treated with cold stress and found that 10 varieties exhibited a significant positive correlation between proline content and cold tolerance, whereas the remaining two varieties showed a negative correlation. This highlights the dependency of proline’s role in plant response to low-temperature stress on factors such as species diversity, stress severity, duration, and other relevant environmental conditions.
Under varying temperatures and durations of low-temperature stress, the chlorophyll content in Dendrobium leaves significantly decreases with prolonged stress duration and decreasing stress temperature. Initially, the change in chlorophyll content is gradual, but as the stress intensifies, the reduction becomes more pronounced, indicating a robust cold-resistance response in the plants. These findings are consistent with research on alfalfa, corn, wheat, roses, poplars, and many other plant species (Du et al. 2024; Jiang et al. 2023; Lainé et al. 2023; Liu et al. 2017; Zhang et al. 2020). Furthermore, correlation analysis reveals a highly significant negative correlation between the variation pattern of chlorophyll content and LT50 in Dendrobium. This suggests that chlorophyll content can be considered a key factor in evaluating the cold resistance of Dendrobium.
The response of ornamental plants to low-temperature stress involves a highly intricate process that warrants deeper exploration. The impact of such stress on the growth and development of ornamental plants is diverse and depends on variables such as plant variety, geographical location, and stress severity. Physiological mechanisms underlying plant resistance to low-temperature stress manifest through varied pathways, encompassing photosynthetic responses, osmotic regulation, scavenging of reactive oxygen species, and intricate ultrastructural changes in leaves. Despite progress in mitigating low-temperature stress in plants, further comprehensive studies are warranted due to the multifaceted interplay of environmental factors. Understanding the effects and interactions of compound environmental factors is pivotal for identifying and selecting varieties equipped with robust cold resistance.
Conclusions
The study demonstrated that low-temperature stress significantly impacts physiological indicators like MDA, free proline, chlorophyll, and yellow leaf rate in Phalaenopsis-type Dendrobium. These indicators exhibited marked changes under stress, correlating strongly with LT50, thereby highlighting the variance in cold tolerance among different Dendrobium varieties. This system enables a comprehensive and effective assessment and comparison of cold tolerance across Dendrobium varieties, enhancing the understanding and management of these plants in cold environments.
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