The Effects of Sudden Freezing on the Biochemical Status of Bamboo Leaves: A Case Study on Nine Species on a Subtropical Plateau

Abstract

The differences in the response of the leaves of different bamboo types to sudden snowfalls in winter were analyzed in order to provide scientific references for the introduction and cultivation of cold-resistant bamboo species and to provide new theoretical information on bamboo afforestation and disaster reduction. A total of nine bamboo species were selected to analyze the physiological and chemical changes in the leaves caused by a sudden snowfall. The results showed that sudden snowfall in winter led to a decrease in the moisture, soluble sugar, and starch contents in the leaves of all of the bamboo species analyzed, but there were increases in the contents of proline, MDA, and H₂O₂ and in the ratios of AsA/DHA and GSH/GSSG. Both the enzymatic activities (SOD, POD, CAT, APX, DHAR, GPX, and GR) and non-enzymatic antioxidant contents (AsA and GSH) were increased after the snowfall, which indicated that the sudden snowfall caused an increase in the antioxidant abilities in the leaves of all bamboo species analyzed. Different bamboo species adopted different strategies for resisting the freezing damage caused by the sudden snowfall. The prevention and mitigation of snow disasters in winter can be scientifically carried out in bamboo forests according to their tolerance abilities.

1. Introduction

Bamboo plants are widely used in tropical and subtropical countries because their shoots are delicious and edible, and their culms are strong and sturdy, making them suitable for building, weaving, papermaking, chopsticks, furniture, etc. Hence, bamboo is one of the most important forest resources because of its fast growth [1]. At present, the bamboo industry plays an irreplaceable role in China’s economic and social development [2]. Bamboo plants mostly prefer a warm and humid climate, but in recent years, the increasing sudden changes in the climate have posed a great threat to bamboo plantations. In early 2008, southern China suffered severe freezing damage with continuous cold rain and snow, which is historically rare, and this brought huge economic losses to bamboo farmers [1]. Therefore, it is of great significance to explore the influence of low temperatures on the physiology of bamboo leaves and the differences in resistance to cold damage in different bamboo species.

Low-temperature stress has a serious effect on the growth and development of plants, and plants adaptively respond to low-temperature stress by regulating their own defense systems [3]. Previous studies have shown that sympodial bamboos require high temperatures during their growth and development; thus, their shoots usually germinate in summer or autumn, and scattered bamboos have shown greater cold resistance than that of sympodial bamboos [4]. Bamboo species with high cold resistance usually show strong antioxidant enzyme activity, a high soluble sugar content, and a high relative content of unsaturated fatty acids [5]. Hu et al. measured some physiological indicators of Bambusa emeiensis and Bambusa multiplex cv. Alphonse-karri under low-temperature stress, such as the SOD and POD activities and the contents of soluble sugar, proline, etc.; in addition, they considered that the contents of soluble protein and proline and the activities of SOD and POD could be used as indicators to evaluate cold resistance in bamboo [6]. Xu also reported that the SOD and POD activities of leaves of bamboo with strong cold resistance were higher than those of bamboo with weak cold resistance, and the leaves of bamboo with stronger cold resistance showed lower MDA contents but higher soluble sugar contents than bamboo with weak cold resistance did [1]. Liu et al. found that the cytoplasmic membrane permeability and MDA content of the leaves showed an upward trend, and the activities of SOD and POD also showed a similar trend with the gradual decrease in external temperature, but the ranges of increase were different among bamboo species [7]. Teng noticed that under natural conditions, the higher the proline content was, the stronger the bamboo’s cold resistance was [8]. Gao reported that bamboo species with strong cold resistance showed low electrical conductivity but a greater increase in proline content and a small change in moisture content [9].

It has been widely recognized that an unexpected sudden snowfall can lead to severe freezing damage in plants, especially in tropical and subtropical regions. However, research on the physiological effects of sudden snowfalls in such regions is limited. Kunming, a city located in Yunnan Province and situated at a subtropical latitude in a monsoon climate zone, often experiences unexpected snowfall events during the winter months every 3–5 years. For instance, a two-day heavy snowfall in Kunming in November 2013 had a significant impact on the growth of garden plants in this area [10]. This particular disaster was both intense and widespread, posing a significant threat to the growth of bamboo species [10]. Therefore, it is crucial to analyze the physiological responses of different bamboo species to unexpected snowfall events.

Phyllostachys species are widely distributed in China; Phyllostachys nigra is a famous ornamental bamboo species, while Phyllostachys mannii and Phyllostachys edulis are extensively used for agricultural tools and in industry [11,12]. B. emeiensis and B. multiplex are high-quality raw materials for papermaking and are mainly distributed in the southwest of China [11,13]. Fargesia species are mainly distributed in the mountains of Southwest China and are highly valued as food [11]. The species of Dendrocalamus are the most abundant in Yunnan, China; the shoots of Dendrocalamus brandisii and Dendrocalamus hamiltonii are unique to Yunnan Province. Their shoots can be eaten fresh and are highly valued as food [11]. The Phyllostachys species are mainly distributed in temperate and subtropical areas, while the Bambusa species grow well in tropical and subtropical areas, and Fargesia bamboo plants are native to mountainous zones of tropical and subtropical areas [11]. The Dendrocalamus species are mainly distributed in subtropical, tropical moist, and tropical dry zones, and they have shown relatively lower low-temperature resistance in comparison with that of other species. In this study, nine representative bamboo species in Kunming, Yunnan Province, China, were chosen to analyze the effects of sudden snowfall on the physiology of bamboo leaves and the differences in the cold damage resistance of different bamboo species. This is of great significance for the selection of cold-tolerant bamboo species and the development and utilization of the bamboo industry.

2. Materials and Methods

2.1. Plant Materials

Leaf samples were gathered from all bamboo species in the bamboo garden of Southwest Forestry University, Panlong District, Kunming City, Yunnan Province, China (102°10–103°40′ E, 24°23–26°22′ N), before and after a sudden two-day snowfall on 22 February 2022. The leaf samples were gathered from four genera and nine species, namely, Ph. nigra, Ph. mannii, Ph. edulis, B. multiplex, B. emeinsis, Fargesia fractiflxa, Fargesia yunnanensis, D. brandisii, and D. hamiltonii. The origins of all the bamboo species could not be clearly traced because they had been transplanted from different areas for about 40 years. All the bamboos were propagated in the bamboo garden by cutting propagation.

A sudden snowfall was forecasted according to the local weather bureau. On 18 February 2022, the weather was sunny, and the temperature of the sampling site was 5–19 degrees; the control group was sampled on this day. On 21–22 February 2022, there was a sudden snowfall during which the minimum temperature reached −1 °C, and the experimental group was sampled. The ontogenetic status of the leaves taken from the control group and the experimental group was the same for each bamboo species. A total of 20 g of leaves was obtained from 9 culms (1-year-old) of three bamboo clusters of each bamboo species in four directions in the middle of the canopy (each cluster of bamboo served as a biological replication, and 6–7 g was sampled from each cluster). The fresh weight of all samples was directly determined in the field, and the samples were then stored in liquid nitrogen for the subsequent determination of the contents of moisture, soluble sugars, starch, and other physiological indexes. The determination of all physiological indexes was repeated three times.

2.2. Moisture Content Determination

The leaf samples collected were dried at 105 °C for 30 min to inactivate plant tissue cells, and they were subsequently dried at 75 °C until a constant weight was achieved to determine the moisture content.

$$Moisture\; content\; (\%)\; =\; (Fresh\; weight\; -\; Dry\; weight)/Fresh\; weight\; \times \; 100\%$$

(1)

2.3. Determination of Soluble Sugars, Starch, and Proline

The contents of soluble sugars and starch were determined by using the phenol–sulfuric acid method according to the methods described by Glassop et al. and Dubois et al. [14,15]. The samples (1.0 g) were ground into powder in liquid nitrogen and then extracted with 10 mL of deionized water at 70 °C. The extractions were centrifuged at 12,000 rpm for 20 min, and the supernatants were collected for the determination of the soluble sugar content. The sediments were stored at −20 °C to determine the starch content. The free proline content in the leaf tissues was determined by using a colorimetric assay according to the method of Wang et al. [16].

2.4. Determination of Physiological Changes in the Antioxidant Defense System

The content of malondialdehyde (MDA) was determined using the thiobarbituric acid method according to Cai [17]. The hydrogen peroxide (H₂O₂) content was determined using the titanium sulfate color method [18]. The superoxide dismutase (SOD) activity was measured using the photoreduction method with nitrogen blue tetrazolium (NBT) [19]. The peroxidase (POD) activity was measured using the guaiacol method [20]. The catalase (CAT) activity was measured using the potassium permanganate titration method according to Li [21].

The ascorbic acid (AsA) and dehydroascorbic acid (DHA) contents were determined according to the method of Kampfenkel [22]. The contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined according to the method of Griffith [23]. The ascorbate peroxidase (APX) activity was determined according to the method of Nakano et al. [24]. The monodehydroascorbate reductase (MDHAR) activity was measured with reference to Hossain and Asada [25]. The dehydroascorbate reductase (DHAR) activity was determined according to the method of Krivosheeva et al. [26]. The glutathione peroxidase (GPX) content was determined according to the method of Meng [27], and the glutathione reductase (GR) activity was measured according to the method of Grace et al. [28].

2.5. Data Analysis

The range of variation in each physiological index was calculated using the following formula:

$$Variation\; range\; (\%)\; =\; |experimental\; group\; -\; control\; group|/control\; group\; \times \; 100\%.$$

(2)

A data analysis was carried out using Microsoft Excel 2310, and the variance in the data was analyzed using SPSS 26.0. A one-way ANOVA was used to compare the physiological indexes of the bamboo species in the control group and the experimental group. The least significant difference (LSD) test was employed to compare the differences among different species. A difference was considered significant at p < 0.05.

3. Results

3.1. Impact of Sudden Snowfall on Moisture Content and Osmotic Regulation in the Leaves of Different Bamboo Types

The contents of moisture, soluble sugar, starch, and proline in the leaves of nine bamboo species under natural conditions showed significant differences (Figure 1).

Before the snowfall, the leaves of B. emeiensis showed the highest moisture content (48.7%), and the leaves of B. multiplex showed the lowest values of only 37.5% (Figure 1A). Contrary to the moisture content, the soluble sugar content showed the lowest values in the leaves of B. emeiensis, with only 3.7 (mg/g) (Figure 1B). However, the leaves of D. hamiltonii showed the highest soluble sugar content, which could reach up to 8.7 (mg/g). As for the starch storage in leaves, F. fractiflxa showed the lowest content, but Ph. mannii showed the highest values; the values were, respectively, 8.8 (mg/g) and 13.7 (mg/g) (Figure 1C). This result showed a trend opposite to the soluble sugar content in the leaves of F. fractiflxa and Ph. mannii, and this implied that there was a greater amount of starch synthesis in the leaves of Ph. mannii. The accumulation of free proline is a well-known metabolic response of plants to stresses [29]. According to Figure 1D, the lowest proline content was shown in the leaves of F. yunnanensis (54.9 μg/g), while the highest value was shown in the leaves of Ph. nigra (448.4 μg/g).

After the sudden snowfall, the moisture, soluble sugar, starch, and proline contents also changed accordingly in the leaves of the different bamboo species (Figure 1). The lowest moisture content was shown in the leaves of D. brandisii, but the highest values were still shown in those of B. emeiensis (Figure 1A). As for the soluble sugar, starch, and proline contents, it was noticed that the soluble sugar content was the lowest in the leaves of F. yunnanensis but the highest in the leaves of F. fractiflxa. However, the starch content was the lowest in the leaves of Ph. mannii but the highest in the leaves of Ph. nigra. The proline content was the lowest in the leaves of F. yunnanensis but also the highest in the leaves of Ph. nigra. Overall, there were no significant correlations between the soluble sugar and proline contents and low-temperature tolerance among the different bamboo species.

The sudden snowfall caused a significant decrease in the moisture, soluble sugar, and starch contents in the leaves of all the bamboo species (p < 0.01) (Figure 1). Compared to the leaves of all the bamboo species before the snowfall, the moisture content in the leaves of F. yunnanensis and D. brandisii decreased by 66.1% and 66.3%, respectively, which implied that the moisture content in the leaves of these bamboos was more easily affected by the sudden snowfall. The highest ranges of variation in the soluble sugar content and starch content in the leaves were in F. yunnanensis (78.9%) and Ph. mannii (78.6%), respectively. Similarly, the sudden snowfall in winter also caused a significant increase in proline content in the leaves of the different bamboo species (p < 0.01); the proline content increased by 115.3% and 116.6% in the leaves of B. multiplex and D. hamiltonii, respectively.

3.2. Impact of Sudden Snowfall on the MDA and H₂O₂ Content and on the Enzymatic Activities of SOD, POD, and CAT in the Leaves of Different Bamboo Types

The accumulation of malondialdehyde (MDA) indicates damage to the cell membrane [30]. As shown in Figure 2A, the MDA content was the lowest in the leaves of D. brandisii (0.6 nmol/g) but the highest in the leaves of Ph. edulis (1.0 nmol/g). After the sudden snowfall, the leaves of Ph. nigra showed the lowest MDA content (0.9 nmol/g), while those of F. yunnanensis displayed the highest values (1.2 nmol/g). Overall, the sudden snowfall in winter led to an increase in the MDA content in the leaves of all the bamboo species, with the content of MDA increasing more significantly in Dendrocalamus and Fargesia than in the Phyllostachys species.

Before the snowfall, the H₂O₂ contents and the enzymatic activities of SOD, POD, and CAT also varied between the bamboo species (Figure 3). As shown in Figure 2B, the leaves of D. hamiltonii showed the lowest H₂O₂ content, while the leaves of Ph. nigra showed the highest values; the values were 30.6 (μmol/g) and 67.8 (μmol/g), respectively.

The enzymatic activities of SOD were relatively low in the leaves of all the bamboo species before the sudden snowfall in winter; the leaves of F. yunnanensis showed the lowest values (246.6 U/g), while those of B. multiplex showed the highest values (380.7 U/g) (Figure 3A). The POD activities in the leaves showed the lowest values in B. emeiensis but the highest in D. brandisii (Figure 3B). As for the CAT activity, it was lower in the leaves of Ph. nigra (40.7 U/g) but the highest in the leaves of D. brandisii (111.6 U/g) (Figure 3C). Generally, there were no obvious trends in the SOD activities with different bamboo species. The POD and CAT activities were higher in the Dendrocalamus bamboos than in other species.

After the sudden snowfall, the H₂O₂ contents and the enzymatic activities of SOD, POD, and CAT increased in all the bamboo species (Figure 2 and Figure 3). A greater increase in H₂O₂ content was shown in the leaves of D. brandisii and D. hamiltonii, with increases of 18.4% and 20.6% (Figure 2B). It was noticed that the sudden snowfall had more of an influence on the H₂O₂ content in the leaves of the Dendrocalamus bamboos than it did on the other species. Additionally, the snowfall showed greater influences on the Dendrocalamus and Fargesia species than on the Phyllostachys and Bambusa species, according to the variations in SOD activity. The SOD activity increased by 285.1% in the leaves of D. hamiltonii but only by 51.6% in the leaves of Ph. mannii (Figure 3A). As for POD and CAT, it was noticed that the sudden snowfall showed a greater influence on the Bambusa and Fargesia bamboos than on the Phyllostachys and Dendrocalamus bamboos (Figure 3B,C). In general, the sudden snowfall showed a greater influence on the bamboo species of Dendrocalamus, Fargesia, and Bambusa. The bamboos with a low resistance to low temperatures showed greater increases in their SOD, POD, and CAT activities; the species of Dendrocalamus mainly relied on SOD for ROS scavenging because of their low enzymatic activities of POD and CAT.

3.3. Effects of Sudden Snowfall on the AsA–GSH System in the Leaves of Different Bamboo Species

Under natural conditions, there were also variations in the contents of non-enzymatic antioxidants in the leaves of the different bamboo species (Figure 4). As shown in Figure 4A, the leaves of the Phyllostachys and Dendrocalamus species showed higher AsA contents than those of the Bambusa and Fargesia species (Figure 4A); the leaves of B. emeiensis showed the lowest AsA content (0.7 nmol/g), while those of Ph. mannii showed the highest content, reaching 2.6 (nmol/g). As for dehydroascorbate (DHA), it was noticed that the bamboos of Phyllostachys showed higher contents than those of other species, implying that the oxidation of AsA into DHA is increased during snowless winters (Figure 4B).

The reduced glutathione (GSH) content was shown to be higher in the leaves of the Dendrocalamus, Fargesia, and Bambusa bamboos but lower in the leaves of the Phyllostachys bamboos (Figure 4C). The leaves of Ph. mannii were shown to have a value of only 641.8 (μg/g), while those of D. hamiltonii reached 1967.9 (μg/g). However, higher contents of oxidized glutathione (GSSG) were determined in the species of Phyllostachys than in the other bamboo species (Figure 4D). This indicated that more GSH was consumed in the Phyllostachys bamboos.

After the sudden snowfall, the AsA and GSH contents significantly increased (Figure 4A,C), while the DHA and GSSG contents accordingly decreased in almost all the bamboo species (Figure 4B,D). It could also be noticed that both AsA and DHA showed the greatest ranges in variation in the bamboo species of Bambusa and Fargesia (Figure 4A,B). However, for GSH, a greater range of variation was shown in the bamboos of Phyllostachys and Bambusa (Figure 4C). This suggested that different bamboo species might employ different strategies to resist the low-temperature stress caused by snowfall. Additionally, the range of variation in the AsA/DHA ratio showed higher values in the species of Bambusa (Figure 4E), while the range of variation in the GSH/GSSG ratio showed higher values in the Phyllostachys bamboos (Figure 4F). This further indicated that different strategies were adopted by the different bamboo species to resist the stress from the sudden snowfall.

The APX activities in the leaves of the Phyllostachys species were comparatively lower than those observed in the other species (Figure 4G), but their range of variation after the snowfall exhibited the highest values. Conversely, the Fargeisa and Dendrocalamus bamboos displayed the smallest ranges of variation in APX activity within their leaves. Additionally, MDHAR and DHAR had higher activity in the leaves of the Bambusa bamboos compared with those of the other species (Figure 4H,I). Notably, a significant decrease was observed in the MDHAR activity, while the DHAR activity showed a significant increase after the snowfall, suggesting that more DHA might be recycled into AsA through reduction by DHAR rather than via reduction from monodehydroascorbate to AsA by MDHAR.

The GPX activity was higher in the leaves of the Bambusa species and in the leaves of F. fractiflxa (Figure 4J). After the snowfall, the activities increased in all the bamboo species, with a consistently increasing range of variation being observed among the different bamboo species in the following order: Phyllostachys < Bambusa < Fargesia < Dendrocalamus. Regarding GR, the leaves of the Phyllostachys species exhibited higher activity than that in most other bamboo species (Figure 4K). After the snowfall, the GR activity increased in all the bamboo species, indicating an enhanced capacity for the reduction of GSSG to GSH and enhanced ROS scavenging abilities.

4. Discussion

4.1. Effects of Sudden Snowfall on Osmotic Regulation in the Leaves of Different Bamboos

Osmotic substances and plant stress resistance are closely correlated, and stresses, such as low temperatures, can affect the contents of osmotic metabolites in plants [31]. Free proline and soluble sugars are important osmoregulatory substances that can increase solute concentrations in plant cells, thereby reducing the freezing point, preventing excessive cell dehydration, and, ultimately, reducing damage to cells [32]. Proline is considered a potent nonenzymatic antioxidant, and greater proline accumulation can improve plant tolerance against various stresses [33]. An increase in the soluble sugar content of plants is also associated with their starch hydrolysis [34]. The glycolysis process in plants is accelerated under adverse conditions, and more energy is generated for physiological responses to various abiotic stresses [35].

In this study, a sudden snowfall in winter reduced the moisture content in different bamboo species, which was consistent with the results of Liu et al. and Wang et al. [36,37]. During the natural cooling process from mid-November to mid-February of the following year, in the leaves of Iris hexagonus, the moisture content gradually decreased throughout the duration of low-temperature stress, and this was negatively correlated with the degree of freezing damage in plants but positively correlated with their cold resistance [36]. After the sudden snowfall, the moisture content significantly decreased in the leaves of all nine bamboo species, but this decrease was more slight in the bamboo species of Phyllostachys and Bambusa in comparison with those of Dendrocalamus and Fargesia. Wang et al. also believed that under cold stress, the decrease in the relative moisture content of plants with strong cold resistance abilities was often slower than that in plants with weak cold resistance abilities [37]. The high moisture content of plants with a high resistance to low temperatures is beneficial for maintaining their normal physiological activities.

The accumulation of sugars plays a crucial role in the acquisition of cold tolerance in plants [38]. Kang et al. observed that the soluble sugar content increased in wheat leaves during the initial stages of freezing stress but rapidly decreased as the stress progressed [39]. Wang et al. reported that the soluble sugar content increased in the branches of Chaenomeles plants exposed to low-temperature stress during the natural cooling process from May 2017 to January 2018 [40]. Wang et al. also suggested that the increase in soluble sugar content within plant cells was beneficial for protecting them from freezing stress [40]. In this study, it was shown that a sudden snowfall in winter caused a decrease in the soluble sugar and starch contents of the leaves of different bamboo species, which contradicted previous research. This could be because the sudden snowfall inhibited the photosynthetic system in the leaves of all the bamboo species, leading to carbohydrates being consumed in large amounts. Additionally, carbohydrates might have been transported to other parts of the bamboos, such as branches and culms, to protect them from freezing damage, resulting in a trend of decreasing soluble sugar in the bamboo leaves. At the same time, the range of the decrease in soluble sugar content was small, but the starch content decreased greatly in the leaves of the Phyllostachys and Bambusa species, indicating that the activity of starch catabolizing enzymes was higher in these bamboo leaves than in those of the Fargesia and Dendrocalamus species. This suggested that sugar metabolism could respond in a more timely manner to freezing stress in the leaves of the Phyllostachys and Bambusa species than in other bamboo species.

Proline can detoxify excess ROSs, improve osmotic adjustments, and lend protection to cell membranes [41]. The sudden snowfall in winter led to an increase in the proline content of the leaves of all the bamboo species, which was consistent with the conclusion of Huang et al. that a low-temperature treatment caused an increase in the proline content of the leaves of nine hedge plants [42]. The current study showed that although the range of increase in the proline content was higher in the leaves of the Dendrocalamus and Fargesia bamboos than in those of the Phyllostachys bamboos, their proline content was always far lower than that of other bamboo species. Hence, proline played a more important role in resisting the damage from the sudden snowfall in the Phyllostachys bamboos than in the Dendrocalamus and Fargesia bamboos.

4.2. Effects of Sudden Snowfall on the Leaves of Different Bamboo Species in Terms of H₂O₂ and MDA Accumulation and SOD, POD, and CAT Activities

The sudden snowfall caused an increase in the contents of H₂O₂ and MDA, as well as increased activities of SOD, POD, and CAT, in the leaves of all the bamboo species. Low temperatures could result in an enhanced production of ROSs, but plant cells equipped with antioxidant systems are able to scavenge free radicals, peroxides, and other ROSs [38]. Shan et al. reported that 1–5 days of hypothermic stress led to a significant increase in the H₂O₂ content of the young leaves of Brassica oleracea, and varieties with high cold tolerance showed a lower H₂O₂ content than that of varieties with low cold tolerance [43].

The accumulation of H₂O₂ under freezing stress was the driving force behind the activation of antioxidant systems, such as those of SOD, POD, and CAT [38]. Previous studies showed that the activities of SOD, POD, and CAT were increased by cold stress [42,44]. ROS accumulation was determined by the balance between production and scavenging. However, the sudden snowfall upset this balance, and ROSs began to accumulate, exerting a toxic effect on the cells [45]. In this study, we observed that the leaves of the Phyllostachys bamboos displayed low variability in H₂O₂ content and SOD and CAT activities, while those of the Bambusa bamboo species showed the greatest range of increase in POD activity and the smallest range of increase in H₂O₂ content. This suggested that POD played an important role in H₂O₂ scavenging in the Bambusa species, as it could promptly scavenge H₂O₂, thus causing a slight difference in the H₂O₂ content before and after the snowfall. In contrast, the leaves of the Dendrocalamus species showed a greater range of increase in H₂O₂ content and SOD activity but lower POD and CAT activities after the snowfall in comparison with the other species. It is known that SOD is the first enzyme to perform detoxification of ROSs by converting superoxide radicals. SOD catalyzes the conversion of ·O²⁻ into H₂O₂, CAT removes the resultant H₂O₂, and POD is one of the key enzymes responsible for H₂O₂ scavenging during oxidative stress in plants [46]. Hence, although the leaves of the Dendrocalmus species showed higher SOD activities and significant increases in their activities after the snowfall in comparison with the other bamboo species, the low ranges of increase in the POD and CAT activities might not have efficiently eliminated the overproduced H₂O₂, leading to its accumulation in the leaves.

MDA is one of the final products of polyunsaturated fatty acid peroxidation in plant cells, and it is a widely used and reliable marker for determining the degree of injury in a stressed plant [47]. Whether it is biological or non-biological, the greater the damage to plants, the higher the intracellular MDA content [48]. Therefore, MDA content can indicate the extent of plant cells’ exposure to environmental stress [49]. The sudden snowfall led to an increase in the MDA content of the leaves of all the bamboo species, which was consistent with the research results from Liu and Luo et al., who found that treatment at 0 °C for 72 h caused an increase in the MDA content of tomato and strawberry leaves, which indicated that the normal functioning of the cell membrane system was impaired [3,50]. It was also observed that the range of increase in the MDA content was lower in the leaves of bamboo species with strong freezing resistance than in those with weak freezing resistance. Liu et al. also reached a similar conclusion: MDA content increased with a drop in temperature [51]. When the temperature is so low that the cellular defense system is powerless to resist it, the normal operation of the membrane lipid peroxidation system of the cell is destroyed, and the MDA content increases to its maximum. It was because the freezing resistance of the Dendrocalamus species was lower that the MDA content sharply increased in their leaves after the sudden snowfall in comparison with the leaves of the other species.

4.3. Effect of Sudden Snowfall on the AsA–GSH System in the Leaves of Different Bamboo Species

The antioxidant capacity depends on the activity of antioxidant enzymes in different cellular compartments, as well as non-enzymatic antioxidants, such as ascorbic acid (AsA) and glutathione (GSH). The AsA–GSH cycle is an important antioxidant system for scavenging reactive oxygen species in plants. The AsA–GSH cycle can quickly and effectively remove the excessive H₂O₂ accumulated in plants under stress, thereby reducing the degree of membrane peroxidation, protecting the active substances in cells, and improving the stress tolerance of plants [52,53].

Reduced AsA and GSH are crucial antioxidant agents in the AsA–GSH cycle [54]. Not only can AsA and GSH directly quench ROSs, but they also serve as enzyme substrates for ROS elimination. They are significant intracellular ROS scavengers and play a highly significant role in plants’ tolerance to low-temperature stress [55]. DHA and GSSG are the oxidized forms of AsA and GSH, respectively, in the AsA–GSH cycle. Alterations in their levels are directly linked to the efficiency of the AsA–GSH cycle, and they are vital for plants’ resistance to various environmental stresses [56]. As important redox pairs in plants, the AsA/DHA and GSH/GSSG ratios can directly represent the redox state of plant cells. Their dynamic fluctuations participate in numerous vital physiological reactions in plants, and they directly affect plants’ adaptability to environmental stresses [57]. The AsA/DHA ratio is a valuable indicator for gauging the available AsA level, and a higher AsA/DHA ratio indicates a higher AsA content [58]. Ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione reductase (GR) are the primary enzyme components of the AsA–GSH cycle. These enzymes, in conjunction with AsA and GSH, participate in the regeneration of antioxidant substances and ROS scavenging in plant cells [59].

The sudden snowfall in winter increased the levels of AsA and GSH, decreased the levels of DHA and GSSG, and raised the ratios of AsA/DHA and GSH/GSSG in the leaves of all the bamboo species. The activities of APX, DHAR, GPX, and GR also increased, while the activity of MDHAR decreased after the sudden snowfall. This implied that the AsA–GSH cycle was enhanced to defend against the damage caused by the sudden snowfall, but it was not enhanced via the regeneration pathway from monodehydroascorbate (MDHA) to AsA. Shan et al. also found that low-temperature stress increased the contents of AsA, GSH, and GSSG, the ratios of AsA/DHA and GSH/GSSG, and the activities of APX and MDHAR [43]. Liu et al. also reported that low-temperature stress at night not only increased the contents of AsA, GSH, and GSSG but also increased the APX, DHAR, and GR activities of tomato leaves [50]. Han et al. found that the activities of the key enzymes APX, MDHAR, DHAR, and GR and the contents of AsA, DHA, GSH, and GSSG significantly increased in a stressful environment [60]. The enhanced enzyme activity within the AsA–GSH cycle facilitated its rapid and effective maintenance, thereby impeding the accumulation of H₂O₂ induced by abiotic stresses [61].

AsA and GSH are the most abundant soluble nonenzymatic antioxidants in higher plants [62]. APX is a key enzyme in the AsA cycle as it converts H₂O₂ into water through the mediation of AsA as a particular electron donor [63]. In the Phyllostachys species, the low H₂O₂ content and AsA/DHA ratio, as well as the achievement of the highest APX activity in the leaves, showed that H₂O₂ was efficiently scavenged by AsA after the snowfall; hence, a large amount of AsA was oxidized, which caused a lower AsA/DHA ratio than that of the other bamboo species. The lower MDHAR and DHAR activities also inhibited the reduction of MDHA, which further limited the increase in AsA content and the AsA/DHA ratio in the leaves of the Phyllostachys species after the snowfall. GR is responsible for GSH reduction and plays an important role in maintaining the GSH pool [64]. Meanwhile, the low activities of GR and GPX and the achievement of the highest GSH/GSSG ratio suggested that the GSH–GSSG cycle did not play a key role in ROS scavenging or resistance against freezing damage caused by the sudden snowfall in the Phyllostachys leaves (Figure 5). Additionally, our previous studies showed that the anatomical characteristics of Phyllostachys leaves significantly contributed to their cold tolerance [65].

DHAR plays a crucial role in regulating cellular AsA redox homeostasis by regenerating AsA from its oxidized state, which is essential for tolerance to various abiotic stresses [35]. After the sudden snowfall, the Bambusa species exhibited significantly higher DHAR activity than APX activity in their leaves, thus limiting the ROS scavenging capacity of AsA. Consequently, more DHA was reduced to AsA during this period, thus further increasing the AsA/DHA ratio in the leaves. The enhanced GR activity promoted GSH production and facilitated the detoxification of H₂O₂ through GPX catalysis. Despite the significant increases in both AsA content and DHAR activity after the snowfall, the decreased APX activity hindered the ROS scavenging abilities of AsA, leading to a further increase in the AsA/DHA ratio. Therefore, the elevated GSH content and GPX activity indicated that the GSH–GSSG cycle played a pivotal role in ROS scavenging in the leaves of the Bambusa species. Similarly, the Fargesia and Dendrocalamus bamboo species also showed high GR and GPX activities and low APX activity, suggesting that the GSH–GSSG cycle has an important role in ROS scavenging. However, the limited GR activity and low APX activity observed in the Dendrocalamus species restricted their potential for GSH regeneration.

5. Conclusions

By analyzing the changes in a series of physiological and biochemical indicators in the leaves of nine types of bamboo after a sudden snowfall, it was found that this unexpected winter snowfall led to lower levels of moisture, soluble sugar, starch, DHA, and GSSG but higher levels of proline, MDA, H₂O₂, AsA, and GSH. Additionally, there was a significant increase in the AsA/DHA and GSH/GSSG ratios. The activities of antioxidant enzymes such as SOD, POD, CAT, APX, DHAR, GPX, and GR also increased. This indicated that the ROS scavenging system was strengthened to resist damage caused by the sudden snowfall. The impacts of the sudden snowfall varied among the different bamboo species, with Phyllostachys being the least affected, followed by the Bambusa species. However, Fargesia and Dendrocalamus were greatly impacted by the winter snowfall. Proline played a more significant role in resisting damage from the sudden snowfall in the Phyllostachys bamboos than in the other species. The different bamboo species employed different strategies for scavenging ROSs, with Dendrocalamus relying mainly on SOD, while Bambusa and Fargesia relied more on POD and CAT for ROS scavenging after the sudden snowfall. In comparison with its role in the Phyllostachys species, the GSH–GSSG cycle played a more important role in ROS scavenging in the Bambusa, Fargesia, and Dendrocalamus leaves.