Cold Acclimation Affects Physiological and Biochemical Characteristics of Betula platyphylla S. under Freezing Stress

: Cold and freezing stress is one of the most harmful environmental stresses, especially in temperate and subtropical areas, that adversely affects plant growth, development, and yield production. Betula platyphylla Sukaczev, also known as white birch, is one of the most valuable, important, and widely distributed tree species in East Asia. This study explored the effects of cold acclimation (CA) in reducing the destructive effect of freezing stress in B. platyphylla seedlings. We measured the physiological and biochemical characteristics of B. platyphylla seedlings, such as chlorophyll content, electrolyte leakage (EL), malondialdehyde (MDA), antioxidant enzymes (such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), and proline content before and after freezing stress to observe the contribution of CA in reducing the detrimental effects of freezing stress. The results showed that CA increased physiological and biochemical characteristics of B. platyphylla seedlings before and after freezing stress, except for chlorophyll content. Antioxidant enzymes were signiﬁcantly positively correlated with proline, MDA, and EL content, and negatively correlated with chlorophyll content. Moreover, histochemical detection (H 2 O 2 and O 2 − ) and cell death were revealed to be induced by cold stress in B. platyphylla seedlings. Furthermore, it was revealed that increased time and decreased temperature of the CA process signiﬁcantly inﬂuenced the physiological and biochemical parameters. Overall, the CA process signiﬁcantly reduced the detrimental effects of freezing stress compared to the control treatment in B. platyphylla seedlings. Taken together, these ﬁndings provide beneﬁcial information toward understanding the mechanism of CA and freezing stress in B. platyphylla . Furthermore, the substantial activity of physiological and biochemical results could be used as selection criteria for screening time and temperature points of cold/freezing stress in further omics analyses. In addition, the combination of current study results, further omics analyses, and genetic engineering techniques directly contribute to sustainable forest management systems, tree plantations, and conservation of tree species, especially non-cold/non-freezing tolerant tree species.


Introduction
White birch (Betula platyphylla S.) is a revolutionary forest plant in northeastern China [1], which is one of the most valuable, important, and widely distributed tree species in east Asia, mostly in China, Korea, and Japan [2]. Betula platyphylla has a rapid growth rate, excellent material properties, high artificial planting survival rate, and a wide range of applications in the manufacture, pharmacy, and landscaping of furniture [1,[3][4][5]. In recent years, several research contributions of B. platyphylla under abiotic stresses can be found, such as drought stress [6,7], salt stress [8], ozone stress [9], heat stress [10], and cold stress [11,12], yet the knowledge is still inadequate and needs to be further explored. The growth of B. platyphylla is compromised due to various biotic and abiotic factors, and cold is among the prevalent constraints. Although cold stress has been studied in B. platyphylla, there have been few studies, which results in a knowledge gap and limited understanding of this cold-tolerant tree species.
Over the past ten years, studies about cold acclimation (CA) have increased significantly [13]. Freezing stress disturbs plant growth and limits plant yield production [13][14][15]. So far, the principal mechanisms of CA in reducing freezing stress effects in plants are complex and still not well understood [16,17]. It was specified that cold or freezing stress affects the molecular, physiological, and biochemical traits [18,19]. Physiological and biochemical markers such as chlorophyll contents, electrolyte leakage (EL), malondialdehyde (MDA), antioxidant enzymes, and proline may be considered as excellent criteria for plant tolerance evaluation [20,21]. EL is an important part of the plant's response to stress. EL caused by stress is often followed by the generation of reactive oxygen species (ROS) and often leads to programmed cell death (PCD) [22]. Furthermore, high levels of ROS, caused by cold stress, result in chloroplast changes and consequently decrease the photosynthesis rate. Plants are protected from damage by antioxidant and non-antioxidant enzymes removing ROS [23]. Besides, proline acts as a water-soluble material with low molecular weight and is considered an effective osmotic regulatory chemical in plant abiotic stress response [20].
As one of the most harmful abiotic stresses, cold stress encourages researchers to examine the various aspects, such as genomics, transcriptomics, and proteomics, in order to produce cold-tolerant species [17,24]. Hence, it was essential to investigate the effects of CA in B. platyphylla to further clarify the ability of this species in resisting freezing stress. CA is the best strategy for plants to survive in winter cold [25]. Studies have revealed that CA significantly elevates membrane stability and integrity of plants under cold stress [16]. Different time and temperature points of the CA process have been reported to increase freezing tolerance in Olea europaea L. [26], Lolium perenne L. [27], and Hordeum vulgare L. [28]. In the present study, we measured the physiological and biochemical parameters, including EL, chlorophyll content, proline, MDA, and antioxidant enzymes, to test the hypothesis that CA reduces the destructive effects of freezing stress in B. platyphylla. In addition, we also measured O 2 •-, H 2 O 2 , and cell death under cold stress to amplify the effect of cold stress in B. platyphylla. These results provide a basis for insights and understanding the physiological and biochemical response of B. platyphylla under cold exposure at different time and temperature points and followed by freezing stress. In addition, this study will be useful to find new methods in the future for B. platyphylla improvement programs.

Materials and Methods
The wild type (WT) seedlings of white birch (Betula platyphylla Sukaczev) were grown on solid agar medium with woody plant medium (WPM), complemented by 0.8 m L −1 6-benzylaminopurine (BA) and 0.02 mg L −1 naphthalene-acetic acid (NAA) in tissue culture bottles. Plants were cut and grown on 0.2 mg L −1 NAA 0.5 Murashige and Skoog (0.5 MS) medium with 1% sucrose and 0.75% agar (pH 6) (16-h light and 8-h dark photoperiod) when the adventitious buds grew up. At 1-month-old, seedlings in 1 2 MS medium were transplanted into a 45-plug tray (3 cm in diameter by 3 cm in height). The seedling growth substratum was black soil (v/v):perlite:vermiculite (4:2:2) and maintained in a growth room at 24 ± 1 • C with a 16-h light and 8-h dark photoperiod. One-month-old plants in the plug tray were moved into an artificial climate box based on the treatment time and temperature points. Plants were divided into five groups; one group was maintained at 24 ± 1 • C as control. The other four groups were subjected to low temperature (LT) treatments, and each group had the stress repeated three times. Four 45-plug tray seedlings were transferred to a temperature of 8 • C and 15 • C for 1 and 2 weeks in the same light and photoperiodic conditions, followed by −10 • C for 10 min. The first until fourth leaves were sampled after treatment for the next measurement. The leaf samples were immediately frozen in liquid nitrogen and stored at −80 • C until use. Three independent biological samples for each treatment were harvested, and each replicate contained ten plants.

Electrolyte Leakage
Electrolyte leakage was measured as follows: three discs (1 cm 2 ) of fresh leaves were placed into beaker glass containing 30 mL deionized water. Beaker glasses were shaken in a vacuum machine for 30 min to separate EL from tissues and measured by using a conductivity meter TDS 3060 (Shenzhen Kedida Electronics Co. Ltd., Shenzhen, China) (mark as S1). The samples were boiled at 100 • C for 20 min to induce cell rupture, and then final conductivity was measured (mark as S2). All physiological indices were repeated with four biological replicates at each time point. EL percentage was obtained as follows: For histochemical analysis, thirty seedlings were transferred to a temperature of 8 • C for 6, 12, and 24 h in the same light and photoperiodic conditions. The procedures for nitroblue tetrazolium (NBT) and 3,3 -diaminobenzidine (DAB) staining were performed using the method reported by Zhang et al. [29], whilst Evans Blue staining was performed as described by Kim et al. [30]. Each sample contained six harvested leaves, and two independent experiments were performed.

Chlorophyll Pigments Content
Fresh leaves of control and low-temperature treatment were measured according to Lichtenthaler [31]. The chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) were extracted with 2.5 mL of 100% acetone into 2.5 mL of 100% ethanol that already contained 0.05 g plants in the dark tube. The absorbance was measured at 470 nm, 649 nm, and 665 nm in a spectrophotometer (Spectrum instruments, Shanghai, China) after 24 h in a dark tube. The following was determined for Chl a, Chl b, and Car content:

Antioxidant Enzymes
Peroxide (POD), superoxide dismutase (SOD), and catalase (CAT) activity were estimated by using POD, SOD, and CAT Assay Kits (Suzhou Comin Biotechnology Co., Ltd., China), according to the manufacturer's instructions. All test tubes contained 0.1 g plant powder. POD activity was calculated by an increase in absorption at 470 nm due to the formulation of products for guaiacol oxidation [20]. The POD value was obtained as follows: POD and SOD activities were measured by using Spectrum instruments (Shanghai, China), with the absorbance recorded for SOD at 560 nm. The SOD value was obtained as follows: where A is the control test tube value and B is the real test tube value. CAT activity was measured by using a spectrophotometer (PerkinElmer Spectrophotometer, Singapore) with an absorbance of 240 nm (mark as A1), and changes in absorbance were measured after 60 s (mark as A2). The CAT value was obtained as follows:

MDA and Proline Measurement
MDA and proline activity were estimated by using the MDA and Proline assay kit (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China). All physiological indices were repeated with three biological replicates at each time point according to the manufacturer's instructions. The absorbance of MDA was recorded at 532 nm and 600 nm for each symbol, while proline absorbance was recorded at 520 nm. MDA and proline were measured using a Spectrophotometer (Spectrum Instruments Spectrophotometer, Shanghai, China). The MDA value was obtained as follows: The proline value was obtained as follows:

Statistical Analyses
Microsoft Excel 2017 and SPSS 25 statistical software were used for statistical analyses. All data are expressed as mean ± standard error (SE). To test for significant differences between treatments, we used two-way ANOVA with Duncan's multiple range test, and p < 0.05 was considered significant for the 8 • C and 15 • C treatments, respectively. A heatmap was generated with http://www.ehbio.com/html, while the correlogram was plotted using the R 3.5.1 software.

Phenotype/Visible Damage of Leaf
In order to examine the effect of cold and freezing stress on B. platyphylla seedlings, we identified the phenotype/visible damage of all treatments ( Figure 1). Cold or freezing stress is well known to disturb plant growth. The results showed that the difference in temperature and time point significantly inhibited B. platyphylla seedling growth. The increasing of time and the decreasing of temperature gradually affected the phenotype of B. platyphylla seedlings (Figure 1c,d). It was revealed that 2w8d was shorter compared to others. Furthermore, 2w8d seedlings had a red stem and yellow leaves ( Figure 1b). This suggests that low temperatures and long exposures interfere with seedling growth, causing stunted, red stems, and yellow leaves/chlorosis. Freezing stress phenotype results showed that all seedlings were dry three days after freezing stress for ten minutes (Figure 1d).

Electrolyte Leakage and MDA Activity
Meanwhile, we further detect the physiological responses of the plant under cold and freezing stress. The effects of CA on EL and the MDA value of B. platyphylla under freezing stress were analyzed systematically ( Figure 2). The results showed that EL in all treatments before freezing stress was higher than that after freezing stress. Meanwhile, the EL value was significantly increased at one week cold-acclimated and non-acclimated B. platyphylla seedlings after freezing stress. The maximum value of EL was noticed at 8 • C for 2 weeks and followed by −10 • C for ten-minute-treated plants compared to all treatments. Furthermore, the level of MDA content was significantly influenced by the duration of CA. Moreover, the MDA content in the B. platyphylla seedlings reached its maximum level with the freezing stress for ten minutes.  We also identified H 2 O 2 , O 2 •-, and cell death by using DAB, NBT, and Evans Blue staining, respectively ( Figure 3). H 2 O 2 , O 2 •-, and cell death are known for their key role in plants under stress conditions. The result showed that CA significantly increased the cell death and ROS in B. platyphylla. According to Figure 3, it was shown that the coloring of the four types of staining was the same in control leaves. However, after 6 h of cold stress, the coloration of all three stainings increased. Among three time points, the 24 h leaves were the darkest, and the 6 h leaves were the lightest, indicating that the long duration of cold stress led to an elevation in the accumulation of O 2 − , H 2 O 2 , and cell death in the leaves. This result suggested that the long duration of cold stress significantly elevated O 2 − , H 2 O 2 , and cell death accumulation in B. platyphylla seedlings.

Changes in Chlorophyll Contents
The direct impact of the CA process and freezing stress in plant growth is in decreasing chlorophyll contents. The average chlorophyll contents for each time and temperature before and after freezing stress showed a different trend (Figure 4). Chl a, Chl b, and carotenoid content of control plants before freezing stress was the highest among all (10.34 mg/g, 3.96 mg/g, and 1.64 mg/g, respectively) and significantly reduced after freezing stress. Interestingly, the Chl a, Chl b, and carotenoid contents of plants after freezing stress in the 1w15d treatment seedlings were higher than before freezing stress. The result was similar to Carotenoid content for 2w15d and 1w8d treatment after freezing stress, which is higher than before freezing stress. On the other hand, Chl a, Chl b, and carotenoid contents of the 2w8d treatment after freezing stress were lower than before freezing stress. Chl a/Chl b content maximally increased in the 2w15d treatment freezing stress (4.35).

Changes of Antioxidant Enzymes
Changes in antioxidant enzymes of the various time points at 8 • C, 15 • C, and −10 • C are shown in Figure 5. The SOD contents increased in the control and 1w15d treatment after freezing stress compared to other treatments. Contrary to SOD activity, the CAT and POD contents continuously increased with the long period of cold and freezing stress. All-time points in POD and CAT measurements were considered significantly different from control. The POD and CAT activities at 8 • C and 15 • C for one and two-week treatments were also significantly increased after freezing stress for ten minutes. Overall, we found the POD and CAT results were significantly different for all assays compared with the control (Figure 5).  . Changes in antioxidant enzymes and proline activity in response to cold and freezing stress. Each value is the average ± standard error (±SE). Different letters indicate a significant difference between 8 • C and 15 • C for one and two weeks at p < 0.05. (a,b); the first letter means the significant difference between time, and the second letter means the significant difference between temperature in a different time and temperature point treatments. 1w15d, 1 week in the icebox with temperature 15 • C; 1w8d, 1 week in the icebox with temperature 8 • C; 2w15d, 2 weeks in the icebox with temperature 15 • C; 2w8d, 2 weeks in the icebox with temperature 8 • C.

Changes of Free Proline Content
Similar to antioxidant enzyme trends, proline content increased in all treatments. Furthermore, the highest amount of proline contents before freezing treatment was in the 2w8d treatment, whereas the lowest proline content was in the control ( Figure 5). Meanwhile, the trend of proline content after freezing stress in control seedlings significantly increased compared to before freezing treatment, illustrating that CA significantly affects the survival of B. platyphylla under freezing stress. From this analysis, the results clearly show that proline content increased with increasing cold stress duration and with the decreasing temperature compared to control before freezing treatment. Furthermore, we showed the relationship among temperature, time points, and physiological and molecular parameters ( Figure 6). It was revealed that the increase of time and decrease of temperature significantly increased the biochemical parameters. In addition, CA significantly influences the survival of plants in freezing conditions.

Plant Physiological Index Relationships
After the CA process (Figure 7), it was found that antioxidant enzyme contents were significantly positively correlated with proline, MDA, and EL content, and negatively correlated with chlorophyll content. Similar to antioxidant enzymes, proline and MDA were also negatively correlated with chlorophyll content. This result illustrated that chlorophyll contents were negatively correlated with all measured physiological and biochemical characteristics of B. platyphylla seedlings under cold and freezing stress. week in the icebox with temperature 15 • C followed by freezing stress; 1w8d_a, 1 week in the icebox with temperature 8 • C followed by freezing stress; 2w15d_a, 2 weeks in the icebox with temperature 15 • C followed by freezing stress; 2w8d_a, 2 weeks in the icebox with temperature 8 • C followed by freezing stress; Control_b, control treatment before freezing stress; 1w15d_b, 1 week in the icebox with temperature 15 • C before freezing stress; 1w8d_b, 1 week in the icebox with temperature 8 • C before freezing stress; 2w15d_b, 2 weeks in the icebox with temperature 15 • C before freezing stress; 2w8d_b, 2 weeks in the icebox with temperature 8 • C before freezing stress (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Discussion
Cold/freezing stress is one of the most harmful environmental stresses encountered by plants [32]. Cold/freezing stress influences all facets of the cellular functions of plants. The detrimental effects of cold stress on plant growth have long been intensively studied by researchers. Changes in physiological and biochemical processes in B. platyphylla seedlings are obvious responses against cold stress. Several pieces of evidence were verified by Wu et al. [12], who claimed that cold stress made the growth rate decrease and contributed to serious effects on B. platyphylla growth. In the present study, the growth of B. platyphylla seedlings was adversely affected when treated at different time and temperature points. The morphological appearance showed that cold treatment has an effect on B. platyphylla seedling appearance, such as dwarfism/stunted, red stem, chlorosis, and withered (Figure 1). There are many reports available for cold stress suggesting it causes plants to be stunted, withered, reddish, and have chlorosis [21,33]. CA significantly increases freezing tolerance through membrane stability and integrity [25]. However, the freezing treatment phenotype result showed that CA only slightly enhanced freezing tolerance (Figure 1d), illustrating that a drastic drop in temperature (8 • C/15 • C to 10 • C) caused severe cell membrane destruction in B. platyphylla. A similar situation was also revealed by Bocian et al. [27] in their previous research implemented on Lolium perenne.
In this study, we further investigated EL content under CA and freezing stress. The results showed that EL was increased with the increasing time and decreasing temperature ( Figure 2). EL is an indicator of cold stress, which causes damage to the plasma membrane. Consistent with previous research, it was revealed that B. platyphylla seedlings had tolerance to protect the cell membrane by avoiding cold damage. After one week of treatment, the cell membrane became more damaged, and the EL value gradually increased, 54.88% and 61.93% in 2w15d and 2w8d, respectively. A similar trend was also represented by Liu et al. [34] in Avena nuda under cold stress. Furthermore, the sudden freezing stress caused injured leaves and showed a drastic elevated EL in control, 1w15d, and 1w8d plants. This result suggested that the short duration of CA significantly affected the cell membrane destruction caused by freezing stress. Freezing stress might significantly increase the damage of the cell membrane. MDA content, together with EL, is a basic parameter to represent and examine the lipid peroxidation degree and the damage of cellular membranes under stress [35,36]. Our result showed that MDA content increased after the CA process (Figure 2). MDA and EL had a slight increase one week after cold stress but significantly increased after freezing stress. Previous research in Brassica napus [37] and Oryza sativa [36,38] also revealed that MDA content increased under chilling and freezing stress, indicating that cold stress-induced ROS accumulation consequently caused lipid peroxidation and disturbed membrane integrity.
The chlorophyll contents represent plant photosynthesis strength [39]. Chlorophyll and carotenoids are the main pigments in the leaf tissue that absorb light energy. As shown in Figure 4, the maximum reduction of chlorophyll contents in the leaves of B. platyphylla seedlings was recorded in control plants that were directly treated with freezing stress for ten minutes. These results are consistent with previous studies representing the reduction of photosynthetic pigments that illustrate an obvious sensitivity of plants to cold and freezing stress [34,36,40]. The reduction of chlorophyll contents was assumed to be the consequence of chlorophyll destruction [41]. The decrease of chlorophyll contents, as an impact of water deficiency, resulted in increased ROS production and consequently led to lipid peroxidation. Several studies have confirmed that the overproduction of H 2 O 2 and O 2 •significantly influences the chlorophyll contents of plants under abiotic stress [42]. Studies conducted by Zhao et al. [36] confirm that ROS accumulation damages the molecular membrane and chlorophyll contents of O. sativa under low-temperature stress. The chlorophyll destruction/degradation changes the green color of leaves to yellow [41]. Cui et al. [43] stated that a freezing temperature (−19 • C) contributed to the reduction of Chl a and Chl b contents in O. sativa. Chl a/b is an indicator for adapting to stress in plants [44]. In our experiment, the Chl a/b value significantly increased in the control after freezing stress, while the Chl a/b value of 1w8d, 1w15d, 2w8d, and 2w15d were not changed significantly after freezing stress. This is because the value of Chl a and Chl b for control seedlings decreased significantly (5.96 mg/g, 1.95 mg/g, respectively) after freezing treatment. Overall, our results showed that there was an inhibitory effect on chlorophyll synthesis under cold stress, both due to the time and decreasing temperature. This statement was also revealed by Huang et al. [45], who claimed that the Chl a and Chl b trend was initially increased, decreased, and finally increased, respectively. We assume that B. platyphylla has the same process in facing cold/freezing stress. Furthermore, the reduction of chlorophyll contents might be related to the inhibition of chlorophyll biosynthesis, which is influenced by chloroplast biogenesis. The inhibition of chlorophyll biogenesis led to the declined granum lamellae and thylakoid membrane proteins [36]. Zhao et al. [36] also revealed that cold stress could inhibit thylakoid protein synthesis.
Another adaptive mechanism observed in B. platyphylla seedlings to cope with cold stress conditions is antioxidant enzyme production. SOD, POD, and CAT are essential enzymes for the plant system, especially under stress [23]. On the other hand, DAB and NBT stained areas showed the accumulation of H 2 O 2 and O 2 •in B. platyphylla seedling's leaves (Figure 3). The accumulation of H 2 O 2 and O 2 •is usually recognized as an effective strategy to defend against stress [46,47]. However, Jamshidi Goharrizi et al. [48] reported H 2 O 2 accumulation increased with increasing cold stress. Similarly, the H 2 O 2 and O 2 •accumulation dramatically elevated with the increasing cold stress reported in Fagopyrum tataricum and Arabidopsis thaliana [49]. The antioxidant enzymes scavenge the ROS to convert H 2 O 2 and O 2 •- [50]. In this study, antioxidant enzymes of B. platyphylla leaves increased after CA treatment. Our results follow previous results from Solanum lycopersicum [51]. This result was also confirmed by Amini et al. [52], who stated that the SOD activity of Cicer arietinum was enhanced under cold stress. As we know, SOD is an essential antioxidant enzyme that is often used for oxidative stress as a biomarker and maintains some stability [45,53]. Within plant cells, SOD reduces the superoxide to H 2 O 2 , which is rapidly decomposed by CAT and POD into O 2 and H 2 O [54,55]. The CAT is found in peroxisomes and decomposes to H 2 O 2 by releasing it from peroxisomal oxidases such as glycolate oxidase in photorespiration [14]. In addition, Omara et al. [56] stated that CAT behavior was determined by the decomposition of H 2 O 2 by CAT, resulting in a reduction of H 2 O 2 . The activity of enzymes can be determined from this reduction. Similar to CA treatment results, the SOD, POD, and CAT activity were also increased under freezing stress. In particular, the SOD, POD, and CAT activity significantly increased after −10 • C treatment compared to plants before freezing stress. The highest activity of these antioxidant enzymes was at 8 • C for two weeks, followed by freezing stress (Figure 5). This indicates that SOD, POD, and CAT play an active role from 8 • C until −10 • C. Moreover, the differences of POD and CAT values on cold-acclimated seedlings compared to control plants after freezing treatment were higher, illustrating that the CA process induced H 2 O 2 production, resulting in elevated antioxidant enzyme activity and antioxidant synthesis as well and alleviating the oxidative stress caused by freezing treatment. This result is similar to Dai et al. [28], who found that antioxidant enzymes are higher in cold-acclimated seedlings compared to non-acclimated seedlings of H. vulgare. Previous studies also clarified that the antioxidant enzyme trend increased on the first day of stress but did not continue and then increased again after a few days. This means that the regulation of the antioxidant enzyme was recidivous. The recidivous trend is the basic response to stress in plants [45].
Proline, as the principal osmotic regulator in plant cells, is believed to play a prominent role in preventing ROS development and in stabilizing the cell structure. Proline is the most important core physiological index for reflecting abiotic stresses [20,57]. In the present study, the proline content was found to be enhanced by the CA process followed by freezing stress (Figure 6). Overall, the proline content of control plants after freezing stress significantly increased compared to before freezing stress (2.5-fold). This result was similar to that obtained earlier [38,58]. In contrast with this result, a negative correlation between proline content accumulation and the CA process was reported in O. europaea [26]. We assumed that the proline content trend was similar to other physiological and biochemical parameters except for chlorophyll contents (Figure 7), illustrating that the increasing of time and decreasing of the temperature of the CA process significantly increased physiological and biochemical parameters.

Conclusions
This study explores how the CA process significantly affects the physiological and biochemical parameters of B. platyphylla seedlings to cope with freezing stress. Specifically, the CA process exposure inhibited plant photosynthesis through chlorophyll degradation, caused increasing ROS production, and consequently led to lipid peroxidation. Furthermore, the physiological and biochemical contents of cold-acclimated B. platyphylla seedlings were significantly different compared to non-cold acclimated B. platyphylla seedlings after freezing treatment, especially for EL, chlorophyll contents, MDA, SOD, and proline content, suggesting that CA can reduce the detrimental effects of freezing stress in B. platyphylla seedlings. Further molecular analyses are needed to strengthen physiological and biochemical analysis results. Taken together, these findings provide beneficial information toward understanding the physiological and biochemical mechanisms of CA and freezing stress of B. platyphylla. Moreover, the substantial activity of physiological and biochemical results could be used as selection criteria for screening time and temperature points of cold stress in further omics analyses. Omics (transcriptomic and proteomic) analysis is potentially used to identify candidate genes that contribute to CA and freezing stress tolerance in B. platyphylla. The identified candidate genes can be used to obtain and insert cold/freezing tolerant plant genes from another tree species through genetic engineering (transgenic plants/CRISPR-Cas9). Consequently, the combination of current study results, further omics analysis, and genetic engineering techniques can directly contribute to increasing sustainable forest management systems, tree plantations, and conservation of tree species, especially non-cold/non-freezing tolerant tree species.