Photosynthesis and Latex Burst Characteristics in Different Varieties of Rubber Trees (Hevea brasiliensis) under Chilling Stress, Combing Bark Tensile Property and Chemical Component Analysis

Abstract

Rubber trees (Hevea brasiliensis) serve as the primary source of natural rubber. Their native habitat is characterized by warm and humid conditions, so they are particularly sensitive to low temperatures. Under such stress, latex burst can cause severe damage to rubber trees, which is due to the uniqueness of their economically productive parts. In order to establish a correlation between young and mature rubber trees and provide a novel prospective for investigating the mechanisms of latex burst and chilling resistance in rubber trees, the chlorophyll contents, photosynthesis, and chlorophyll fluorescence parameters in four varieties of one-year-old rubber tree seedlings were analyzed under artificially simulated chilling stress. The latex burst characteristics were subsequently recorded. A comprehensive statistical analysis of the chilling-resistance rank was conducted using the membership function method and the combination weighting method. Meanwhile, chemical compositions and tensile properties of barks from two-year-old twigs of mature rubber trees were ascertained. A correlation analysis between chilling resistance, chemical compositions, and tensile properties was performed to identify any interrelationships among them. The results showed that the number and the total area of latex-burst positions in variety Reken628 seedlings were greater than those in other varieties, and the lowest number and total area of latex-burst positions were observed in variety RRIM600 and variety PR107, respectively. With the exception of variety GT1, nectar secretion was noted in all other varieties of rubber tree seedlings under chilling stress. The chilling resistance of the four varieties decreased in the following order: variety GT1 > variety RRIM600 > variety PR107 > variety Reken628. The chilling resistance was strongly (p < 0.001) negatively correlated with cellulose content and acid-insoluble lignin content, respectively. The total area of latex burst was significantly (p < 0.001) and positively correlated with holocellulose content and maximum load, respectively. Furthermore, this study also provides new insights into the mechanism of nectar secretion induced by low temperatures and its association with the chilling resistance of rubber trees.

1. Introduction

The rubber tree (Hevea brasiliensis.) is a perennial tree crop native to the Amazon rainforest in Brazil. Rubber trees produce high-quality rubber in commercial quantities, accounting for more than 98% [1] of the world’s natural rubber supply. Despite the fact that rubber tree plantations were constructed successfully between 18° N and 24° N in the south of China on a large scale in the 1950s, chilling injury remains a significant challenge for the rubber tree growing industry, particularly during the winter and early spring seasons [2]. Rubber tree plantations situated on the edge of the tropics and in the southern subtropics of China necessitate the cultivation of cold-resistance rubber trees. Though the rubber tree is chilling-tolerant to a certain extent, it is susceptible to cold damage under conditions of intolerable temperatures or excessive chilling accumulation. Within their respective habitats, various plant species exhibit distinct optimal temperatures for photosynthesis. The optimal temperature for photosynthesis in rubber trees lies between 25 °C and 30 °C. When subjected to temperatures below 10 °C, the photosynthetic activity of rubber trees ceases [2]. The sucrose produced through photosynthesis serves as the unique precursor for rubber biosynthesis in the laticifers [3]. Low temperatures impede all biochemical reactions involved in photosynthesis [4]. Upon cessation of photosynthesis in the rubber tree, the plant maintains its metabolic activity by respiring stored starch and other photosynthetic carbohydrates, despite persistent chilling stress. Moreover, the rubber tree’s inability to synthesize new photosynthetic compounds weakens its cold resistance. This susceptibility is further exacerbated by alterations in pigment composition and shifts in the protein, lipid, and fatty acid profiles of plant cell membranes, as well as variations in the abundance of these chemical components. Consequently, chilling injuries to rubber trees are unavoidable. Exposure to chilling stress can induce leaf necrosis, shriveled branches, and latex bursts throughout the tree. The trunk bark burst resulting from this stress not only leads to bark necrosis but also precipitates secondary disease and pest damage to rubber trees [5,6,7,8]. These factors collectively contribute to a decline in the resistance of rubber trees and an escalation of economic loss.

Rubber latex is synthesized in laticifers, which are differentiated from the cambium. These laticifers form an anastomosed network structure and are arranged in rings parallel to the vascular cambium following maturation [9]. When rubber trees are subjected to chilling temperatures, the outer bark layers cool more rapidly than the xylem and exhibit significant tensile strength in the tangential direction. This leads to cracks forming along the radical surface of the outer bark when exposed to abrupt drops in temperature, causing laticifers to break. The latex then flows between the bark and the xylem, where the pressure is lower, subsequently condensing into colloids. If the expansive force of the latex and colloids is less than the tension that the bark can withstand, the colloids remain within the bark. Conversely, if the expansive force exceeds this tension, the bark bursts, allowing the latex to escape from the tree through fissures [10]. Therefore, the tensile property of the bark is a crucial factor influencing bark burst in rubber trees under chilling stress.

The mechanical properties of plant tissues are largely determined by the arrangement of fibers in cell walls [11]. The cell wall must possess the ability to withstand high tensile stresses, either permitting elastic deformation in response to changes in turgor pressure or providing sufficient extensibility to accommodate elongation [12]. As an active and dynamic organelle, the cell wall is composed of a considerable amount of the polysaccharide cellulose. In addition to cellulose, the primary cell wall also contains a three-dimensional network of hemicellulose, pectin, and glycoproteins. The secondary wall, which originates from the thickening of the primary wall, is typically impregnated with lignin [13]. Plant cell walls exhibit marked variations in composition and structure across different cell types, developmental stages, and species. Thus, the composition and structure of the cell wall can be flexibly modified during various biological processes, in response to different stresses and to meet specific functional requirements [14].

The constitution and structure of the cell wall affect the physical and mechanical properties of plants. Furthermore, changes in cell wall components can impact photosynthetic parameters under abiotic stresses [15]. The economic yield of rubber trees is derived from their bark, which also experiences the latex burst caused by chilling stress. By establishing a correlation between the chemical components of the bark and its physical properties, we can better understand the mechanisms behind the latex burst under chilling stress. Moreover, we can verify changes in bark components among different varieties of rubber trees that affect photosynthetic parameters under chilling stress. In order to ensure the feasibility of the simulation experiment, we utilized one-year-old budded rubber trees to withstand the chilling stress. Twigs from two-year-old mature rubber trees were harvested for the measurement of bark physical properties and chemical component analysis. This study aimed to determine the variations in chlorophyll content, photosynthetic parameters, and chlorophyll fluorescence parameters across different rubber tree varieties under chilling stress. Utilizing the membership function method and the combination weighting method, we determined the order of chilling resistance among four varieties of rubber trees. Notably, the bark latex burst conditions exhibited variations in chilling resistance across these varieties when subjected to chilling stress. We constructed a correlation matrix based on the comprehensive evaluation value of different varieties, considering factors such as their total latex burst areas, their bark physical properties, and their chemical components. The connection between one-year-old budded rubber trees and mature trees was made to reveal the mechanism underlying latex burst in rubber trees under chilling stress.

2. Materials and Methods

2.1. Experimental Design and Treatment

One-year-old budded rubber tree seedlings and mature rubber trees (thirteen years old) were cultivated in the National Tropical Plants Germplasm Resource Center—Rubber Tree. Prior to application of chilling stress, these one-year-old budded seedlings were maintained in the nursery of the same center under natural conditions (temperature range of 23–29 °C), located at coordinates 19°30′50.4″ N, 109°29′31.2″ E, and an altitude of 168.7 m. The varieties of rubber trees examined in this study included PR107, RRIM600, Reken628 and GT1. Seedlings with similar growth characteristics were subjected to chilling treatments (16/4 °C, day/night), under a photoperiod of 12 h, relative humidity of 80%, and light intensity of 100 μm m⁻² s⁻¹. On the 0, 1st, 3rd, and 5th days of the chilling treatment, photosynthetic pigment contents (Chl a, Chl b, T chl, and carotenoid), chlorophyll fluorescence, and photosynthesis parameters were measured in leaves from the same stories. Rubber tree phenotypes were observed daily during the chilling treatment, and the representations of latex burst were recorded from emergence to the termination of the treatment. Healthy two-year-old rubber tree twigs (with 5 plants per variety) exhibiting similar growth were collected for bark tensile property and chemical component measurement.

2.2. Photosynthetic Pigment Content Determination

Photosynthetic pigment contents were determined on the 0, 1st, 3rd, and 5th days of the chilling treatment, following the experimental guidance [16]. Each sample (0.05 g) was placed in a 15 mL centrifuge tube containing a 15 mL intermixture solution (acetone/ethanol/ultra-pure water = 4.5:4.5:1), then incubated in the dark at room temperature for 24 h (shaking several times) until the leaves turned white. Subsequently, absorbance readings of the extract solution were obtained using a UV/VIS spectrophotometer (PGENERAL T6 Newcentury, Beijing, China).

2.3. Chlorophyll Fluorescence Parameter Examination

The parameters of chlorophyll fluorescence were examined on fully expanded leaves from the same storey in rubber trees using a portable pulse-modulated fluorescence instrument (PAM-2500, WALZ, Effeltrich, Germany), according to the operation manual. The rubber trees underwent a 30 min dark adaptation period prior to measuring the initial fluorescence (F_o) with the fluorescence instrument. Following a saturating pulse, the maximum fluorescence (F_m) was obtained, which facilitated the calculation of the potential quantum photochemical efficiency of PSII [F_v/F_m = (F_m − F_o)/F_m]. At the same time, the actual photochemical yield of PSII (Φ_PSII) was determined using the equation Φ_PSII = (F_m’ −₌ F_s)/F_m’, where F_m’ represents the maximum fluorescence of light-adapted leaves and F_s denotes the stable fluorescence.

2.4. Photosynthetic Parameter Measurement

Net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and transpiration rates (T_r) were concurrently measured for leaves from the same storey using a portal photosynthesis system (Li-6400, LI-COR BioSciences, Lincoln, NE, USA). The leaf chamber ensured a controlled environment with a constant photosynthetic photon flux density (PPFD) of 100 μmol m⁻² s⁻¹, CO₂ concentration ranging between 360 and 380 μmol mol⁻¹, and a consistent leaf temperature of 25 ± 1 °C throughout the measurement procedure.

2.5. The Area of Latex Burst Determination

The area of the latex burst was ascertained using an electronic digital caliper. The diameter of the dot-shaped latex burst location and the side lengths of the runner-shaped location were recorded. The area was subsequently calculated based on these data.

2.6. Tensile Property Test

Bark tensile property tests were conducted on dog-bone samples (n ≥ 10) using a universal testing machine (50KN CMT5504, MTS Systems Co., Ltd., Shenzhen, China), with control provided by the Power Test_Dooc software (Power Test_Dooc V3.6, Shenzhen, China), following our previous methodology [17]. The test velocity was set at 1.5 mm min⁻¹ with a gauge length of 200 mm. A minimum of 10 replicates (from 5 plants) were tested for each rubber tree variety.

2.7. Chemical Composition Analysis of Rubber Tree Bark

All the composition analyses were conducted on duplicate samples, following the nitric acid-ethanol and GB/T standard measurement methods. The content of pectin (GB/T 10742-2008) [18], cellulose [19], acid-insoluble lignin (GB/T 2677.8-1994) [20], and holocellulose (GB/T 2677.10-1995) [21] in the samples was determined.

2.8. Statistical Analysis

With the exception of the tensile property test, all experiments were conducted in triplicate. Data analysis was performed using a one-way ANOVA test, with significant statistical differences between mean values of indicators determined by running Duncan’s multiple range test in Excel 2021 at a significance level of p < 0.05. The membership function value of the indicator and the entropy weight of the indicator were calculated using Excel 2021. The Spearman correlation between chilling resistance, latex burst sensitivities, and their physiological and mechanical parameters was constructed using Origin 2021.

Combination weights were ascertained as previously described [22].

The method of the membership function value was employed to conduct a comprehensive evaluation of the chilling resistance across various physiological indicators. The corresponding formulas are as follows:

U(Xij) = (x_max − x_ij)/(x_max − x_min)

(1)

$$\mathrm{Vj}={\overline{\mathrm{x}}}_{\mathrm{j}}/\mathsf{\sigma }\mathrm{j}$$

(2)

$$\mathrm{Wvj}=\mathrm{Vj}/\sum \mathrm{V}\mathrm{j}$$

(3)

$${\mathrm{D}}_{\mathrm{M}}={\sum }_{\mathrm{j}=1}^{\mathrm{n}}\left[\mathrm{U}\left(\mathrm{X}\mathrm{i}\mathrm{j}\right)\mathrm{W}\mathrm{v}\mathrm{j}\right]$$

(4)

where U(Xij) is the membership function value of the j-th comprehensive indicator in variety i, x_ij is the value of the j-th indicator in variety i, and x_max and x_min represent the maximum and minimum values of the j-th indicator, respectively. Vj is the standard deviation coefficient of the j-th indicator, $\overline{\mathrm{x}}$_j is the mean value of the j-th indicator, and σj represents the standard deviation of the j-th indicator. Wvj and D_M stand for the weight of the j-th indicator and the comprehensive evaluation value by the membership function method.

$$\mathrm{Yij}=\mathrm{U}\left(\mathrm{X}\mathrm{i}\mathrm{j}\right)/{\sum }_{\mathrm{i}=1}^{\mathrm{n}}[\mathrm{U}\left(\mathrm{X}\mathrm{i}\mathrm{j}\right)]$$

(5)

$$\mathrm{Ej}=-{\displaystyle \frac{1}{\mathrm{I}\mathrm{n} \mathrm{n}}}{\sum }_{\mathrm{i}=1}^{\mathrm{n}}\mathrm{Y}\mathrm{i}\mathrm{j}$$

(6)

$$\mathrm{Wej}={\displaystyle \frac{1-\mathrm{E}\mathrm{j}}{\mathrm{k}-{\sum }_{\mathrm{j}=1}^{\mathrm{m}}\mathrm{E}\mathrm{j}}}$$

(7)

$$\mathrm{Wj}={\displaystyle \frac{\sqrt{\mathrm{W}\mathrm{v}\mathrm{j}\ast \mathrm{W}\mathrm{e}\mathrm{j}}}{\sum _{\mathrm{j}=1}^{\mathrm{m}}\sqrt{\mathrm{W}\mathrm{v}\mathrm{j}\ast \mathrm{W}\mathrm{e}\mathrm{j}}}}$$

(8)

$$\mathrm{D}=\sum \left[\mathrm{U}\right(\mathrm{X}\mathrm{i}\mathrm{j})\mathrm{W}\mathrm{j}]$$

(9)

where Yij is the weight of the j-th indicator of the i-th variety. The modification of the Yij value refers to reference [22]. Ej represents the information entropy of the j-th indicator. Wej and Wj stand for the entropy weight of the j-th indicator and the combination weight. D is the comprehensive evaluation value obtained by the combination weighting method.

3. Results

3.1. Changes in Photosynthetic Pigment Content under Chilling Stress

The analysis of experimental data revealed that the trends in photosynthetic pigment content varied among rubber tree varieties under chilling stress (Figure 1A–D). Chilling stress resulted in a gradual increase in chl b and carotenoid contents in the leaves of variety GT1 seedlings. However, exposure to chilling stress initially increased (after 1 and 3 days) and then decreased (after 5 days) the photosynthetic pigment contents (chl a, T chl) in these seedlings. During this period, the photosynthetic pigment content in the leaves of variety GT1 seedlings was significantly (p < 0.05) higher than that in the other varieties (Figure 1A–D). After 3 days of chilling stress, the highest chl a and T chl contents were observed in variety GT1, with enhancements of 15.86%, 18.34%, and 16.48%, respectively, compared to pre-stress levels. After 5 days of chilling stress, there was a significant increase in the carotenoid contents of leaves in variety PR107, variety RRIM600, variety Reken628, variety GT1 by 64.47%, 99.14%, 66.77%, and 73.93%, respectively, compared to pre-stress levels (Figure 1D). Following a 5-day period of chilling stress, the increases in chl b content were observed to be 33.02%, 51.22%, 29.67%, and 27.51% for variety PR107, variety RRIM600, variety Reken628, variety GT1, respectively, relative to their pre-stress levels. However, differences in photosynthetic pigment contents in variety PR107 and variety Reken628 were significantly (p < 0.05) lower than those of the other two varieties (Figure 1A–D).

During the chilling stress, the chlorophyll content of variety PR107 showed a trend of first decreasing and then increasing (

Table 1. Changes in chlorophyll contents of each rubber tree variety under different chilling stress days.

Variety	Chilling Stress Time (Day)	Chl a (mg g−1)	Chl b (mg g−1)	T Chl (mg g−1)	Caro (mg g−1)
PR107	0	2.9870 ± 0.0805 ab	0.9472 ± 0.0351 b	3.9341 ± 0.1156 b	0.5353 ± 0.0091 c
	1	2.9182 ± 0.0766 b	0.9198 ± 0.0259 b	3.8380 ± 0.1025 b	0.5340 ± 0.0125 c
	3	3.0843 ± 0.0054 ab	0.9794 ± 0.0021 b	4.0637 ± 0.0071 b	0.5908 ± 0.0002 b
	5	3.1734 ± 0.0412 a	1.2600 ± 0.0166 a	4.4334 ± 0.0578 a	0.8804 ± 0.0119 a
RRIM600	0	2.8712 ± 0.0503 b	0.9370 ± 0.0160 b	3.8083 ± 0.0663 b	0.5124 ± 0.0154 b
	1	3.0186 ± 0.1447 b	0.9794 ± 0.0424 b	3.9980 ± 0.1872 b	0.5464 ± 0.0225 b
	3	3.0179 ± 0.0462 b	0.9533 ± 0.0175 b	3.9712 ± 0.0637 b	0.5758 ± 0.0085 b
	5	3.5649 ± 0.1123 a	1.4169 ± 0.0449 a	4.9818 ± 0.1572 a	1.0204 ± 0.0353 a
Reken628	0	2.9633 ± 0.0925 a	0.9515 ± 0.0289 b	3.9148 ± 0.1213 b	0.5459 ± 0.0289 b
	1	2.4659 ± 0.1003 b	0.8293 ± 0.0349 c	3.2952 ± 0.1351 c	0.4430 ± 0.0167 c
	3	2.5231 ± 0.0477 b	0.8193 ± 0.0157 c	3.3423 ± 0.0634 c	0.5240 ± 0.0107 b
	5	3.1033 ± 0.0969 a	1.2338 ± 0.0384 a	4.3371 ± 0.1353 a	0.9104 ± 0.0288 a
GT1	0	3.9467 ± 0.1118 b	1.3295 ± 0.0410 c	5.2762 ± 0.1525 c	0.6756 ± 0.0264 c
	1	4.1778 ± 0.0923 b	1.4486 ± 0.0326 bc	5.6265 ± 0.1249 bc	0.7087 ± 0.0140 c
	3	4.5727 ± 0.1405 a	1.5734 ± 0.0494 ab	6.1461 ± 0.1898 ab	0.7902 ± 0.0188 b
	5	4.2558 ± 0.0786 ab	1.6952 ± 0.0312 a	5.9510 ± 0.1098 a	1.1751 ± 0.0219 a

Data were represented as means ± SEs. Means that do not share the same letter(s) are significantly different at p < 0.05 by Duncan’s test of one-way ANOVA. Chl a, chlorophyll a content; Chl b, chlorophyll b content; T chl, total chlorophyll content; Caro, carotenoid content.

). After 5 days of stress, except for its chlorophyll a content, the contents of other photosynthetic pigments were significantly (p < 0.05) higher than those before the stress, with the carotenoid content increasing by 64.47% compared to that before the stress. The carotenoid content of variety RRIM600 seedlings showed a continuous upward trend, while the contents of other photosynthetic pigments exhibited an up-down-up trend under chilling stress. After 5 days of stress, its photosynthetic pigment contents were significantly (p < 0.05) higher than those of 0–3 days of chilling stress. Compared with before the stress, the increase in carotenoid content after 5 days of stress was the most pronounced, reaching 99.14%. After 1 day of chilling stress, the contents of chl a, T ch, and carotenoids in variety Reken628 decreased to the lowest values during stress, and the differences were significant (p < 0.05) compared with those before stress. After 3 days of stress, the content of chlorophyll b in variety Reken628 reached the lowest value during stress, but it was not significant (p < 0.05) compared with that after 1 day of stress. After 5 days of stress, all photosynthetic pigment contents in variety Reken628 reached the highest values during stress, except for chlorophyll a, which were significantly (p < 0.05) higher than those before stress. Especially, the content of carotenoid increased by 66.77% compared with that before stress. During the chilling stress process, the content of all photosynthetic pigments in variety GT1 showed a continuous upward trend. After 5 days of chilling stress, except for the chl a content, the differences in other pigment contents compared to before the stress reached a significant (p < 0.05) level. The increase in carotenoid content was the most obvious, reaching 73.93%.

3.2. Effect of Chilling Stress on Fluorescence Parameters in Rubber Trees

After 1 day of chilling stress, the F_v/F_m values of variety PR107, variety RRIM600, variety Reken628, and variety GT1 increased by 11.09%, 4.04%, 14.14%, and 13.72%, respectively, compared to their pre-stress levels. However, these differences were not statistically significant (p < 0.05) among them (Figure 2A). After 3 days of chilling stress, the F_v/F_m value of variety RRIM600 slightly increased compared to that after 1 day of chilling stress, but those in variety PR107, variety Reken628, and variety GT1 slightly decreased by 0.93%, 3.14%, and 1.08%, respectively. In addition, the F_v/F_m value of variety Reken628 was lower than that of the other three varieties after 3 days of chilling stress, as the same statistical result was observed after 5 days of chilling stress. After 5 days of chilling stress, the F_v/F_m values in variety PR107, variety GT1 increased by 7.19% and 6.64%, respectively, while those in variety RRIM600 and variety Reken628 declined by 0.54%, 2.55%, respectively compared with them before chilling stress.

Before the chilling stress, the Φ_PSII value in variety GT1 was notably higher than that in the other three varieties, but significant (p < 0.05) differences were only observed between variety GT1 and variety RRIM600 (Figure 2B). After 1 day of chilling stress, the decrease in Φ_PSII values in variety PR107, variety Reken628, and variety GT1 were 43.73%, 45.53%, and 18.52%, respectively, compared with those before chilling stress. In contrast, the Φ_PSII value in variety RRIM600 increased by 15.29%. The Φ_PSII value in variety GT1 remained higher than that in the other three varieties after 1 day of stress exposure. Furthermore, after 3 days of chilling stress, the Φ_PSII values in variety PR107 and variety Reken628 increased by 20.68% and 16.24%, respectively, while those in variety RRIM600 and variety GT1 decreased by 11.75% and 26.47%, respectively, compared to their values after 1 day of chilling stress, but there were no significant (p < 0.05) differences among them. After 5 days of chilling stress, the Φ_PSII value in variety GT1 was higher than that in the other variety seedlings, with significant differences (p < 0.05) observed between variety GT1 and variety RRIM600.

3.3. Impact of Continuous Chilling Stress on Photosynthetic Parameters in Rubber Trees

The leaf photosynthetic parameters, including the net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular carbon dioxide concentration (C_i), and transpiration rate (T_r) values in variety PR107, variety RRIM600, variety Reken628, and variety GT1 seedlings, significantly decreased under the chilling stress. But these parameters exhibited varying trends (Figure 3). The P_n value in the seedlings declined after 1 day of chilling stress, compared with that before chilling stress. Figure 3A showed that the decline was least pronounced in variety GT1. After 3 days of chilling stress, the P_n value in variety GT1 seedlings gradually increased by 54.76%, whereas the P_n values in variety PR107, variety RRIM600, and variety Reken628 seedlings continuously decreased, accounting for 15.90%, 14.66%, and 7.81%, respectively, compared to their values after 1 day of chilling stress. After 5 days of chilling stress, the P_n values in variety RRIM600 and variety GT1 increased, but those in variety PR107 and variety Reken 628 continued to decrease. There were significant (p < 0.05) differences between variety GT1 and the other varieties. Compared with non-chilling stress, the P_n values in variety PR107, variety RRIM600, and variety Reken628 decreased by 60.02%, 49.72%, and 68.11%, respectively, while the P_n value in variety GT1 reached its maximum.

After 1 day of chilling stress, there was a clear increase in the G_s values of variety RRIM600 and variety Reken628, whereas the G_s values of variety PR107 and variety GT1 decreased (Figure 3B). A rapid decline of the G_s values was visible in variety PR107, variety RRIM600, variety Reken628, and variety GT1 (Figure 3B), accounted by 63.06%, 94.18%, 87.13%, and 67.04%, respectively, after 3 days of chilling stress, compared to the values recorded after 1 day of chilling stress. But there were no significant differences (p < 0.05) among them. The G_s values followed a similar pattern as the P_n values after 5 days of chilling stress. Compared with the G_s values before chilling stress, the values in variety PR107, variety RRIM600, variety Reken628, and variety GT1 were reduced by 74.12%, 79.49%, 85.93%, and 59.49%, respectively. Notably, the value of variety GT1 seedlings was significantly (p < 0.05) higher than that of the others after 5 days of chilling stress.

The C_i values in variety PR107 and variety RRIM600 showed an increasing trend, while those in variety Reken628, variety GT1, demonstrated a decreasing trend. Although the C_i value of variety RRIM600 was higher than the others, significant (p < 0.05) differences were only observed between variety RRIM600 and variety Reken628 after 1 day of chilling stress (Figure 3C). Under chilling stress, the C_i value of the seedlings in all varieties decreased as the duration of the stress increased. However, there were insignificant (p < 0.05) differences among them after 3 days of chilling stress. Compared with the C_i values after 3 days of chilling stress, those in variety RRIM600, variety Reken628, and variety GT1 showed an increasing trend, but those in variety PR107 showed a contrary change after 5 days of chilling stress. Later, the C_i values in variety PR107, variety RRIM600, variety Reken628, and variety GT1 were reduced by 53.67%, 62.35%, 54.20%, and 67.76%, respectively compared with those before chilling stress. There were insignificant (p < 0.05) differences among them.

The T_r values in various varieties followed a similar trend in alteration as the G_s values during the chilling stress phrase (Figure 3D). However, there were no significant (p < 0.05) differences among T_r values in different varieties after 1 day of chilling stress. As the duration of chilling stress was extended, the T_r value in variety GT1 was more pronounced than that in the other varieties. The differences in the T_r values were significant (p < 0.05) for variety GT1 compared to the other varieties after 5 days of chilling stress. Compared with T_r values in variety PR107, variety RRIM600, variety Reken628, and variety GT1 before chilling stress, the T_r values decreased by 62.87%, 69.81%, 79.21%, and 31.49%, respectively, after 5 days of chilling stress. The T_r values in variety GT1 showed a milder decline compared with those in the other varieties.

3.4. Latex Burst and Nectar Secretion Represented the Visual Damage of Chilling Stress

The syndrome of latex burst emerged in rubber tree seedlings after 3 days of chilling stress. Symptomatic seedlings exuded a small amount of latex on their petioles (Figure 4(A-1, A-2, B-1, C-1, D-1)), tender stems (Figure 4(B-2, C-2, C-3, D-2, D-3)), and leaf scars (Figure 4(C-2)). This was accompanied by the appearance of dark spots on the leaves (Figure 4(B-3)). The statistical data presented in Table 1 revealed variations in the manifestations of latex burst among different varieties. A stripped latex burst was evident in Reken628 (Figure 4(C-2)). Furthermore, the total area of latex burst in variety Reken628 seedlings was the most extensive, and the total number of latex-burst positions was the highest (

Table 2. Damage observation and data statistics of latex burst in rubber tree seedings after chilling stress.

Variety	NDP	NSP	Total Number	Area of DP (mm2)	Area of SP (mm2)	Total Area (mm2)	Location
PR107	6	0	6	14.2874	0	14.2874	Petiole
RRIM600	4	0	4	32.9117	0	32.9117	Petiole, stem
Reken628	8	1	9	35.9893	12.9428	48.9321	Petiole, stem, leaf scar
GT1	6	0	6	32.9124	0	32.9124	Petiole, stem

NDP, number of dotted latex-burst positions; NSP, number of stripped latex-burst positions.

). Interestingly, with the exception of variety GT1, the nectaries in all other varieties of rubber tree seedlings secreted nectar as the duration of chilling stress increased (Figure 4(A-3, B-4, C-4)).

3.5. Different Tensile Properties in Barks of Different Rubber Tree Varieties

In total, six variety PR107 bark specimens, twenty two variety RRIM600 bark specimens, eight variety Reken628 bark specimens, and seven variety GT1 bark specimens were successfully tested and analyzed. The bark thickness of two-year-old twigs across these varieties is illustrated in

Table 3. Tensile properties (bark thickness, maximum load, tensile strength, elongation at break point, Young’s modulus) of barks in different rubber tree varieties.

Variety	BT (mm)	ML (N)	TS (MPa)	EBP (%)	YM (MPa)
PR107	0.7383 ± 0.0577 b	6.7783 ± 0.7080 b	1.9400 ± 0.2762 b	3.2861 ± 0.4361 b	59.5229 ± 4.7126 a
RRIM600	0.8005 ± 0.0256 b	9.8736 ± 0.5952 a	2.5005 ± 0.1692 ab	4.5051 ± 0.3424 b	72.7703 ± 16.4655 a
Reken628	0.9875 ± 0.0706 a	10.7900 ± 1.3989 a	2.2237 ± 0.3224 ab	4.7181 ± 0.7977 b	54.1421 ± 8.2707 a
GT1	0.7643 ± 0.0452 b	10.5743 ± 0.9489 a	2.7643 ± 0.1917 a	7.1811 ± 0.8670 a	42.8443 ± 6.8711 a

Data were represented as means ± SEs. Means that do not share the same letter(s) are significantly different at p < 0.05 by Duncan’s test of one-way ANOVA. BT, bark thickness; ML, maximum load; TS, tensile strength; EBP, elongation at break point; YM, Young’s modulus.

. It can be seen that Reken 628 exhibited the significantly (p < 0.05) highest bark thickness (BT) value compared to the other varieties. Among them, the maximum load (ML) was observed to follow an order of variety Reken628 > variety GT1 > variety RRIM600 > variety PR107 with values of 10.7900 N, 10.5743 N, 9.8736 N, and 6.7783 N, respectively. It is important to note that the maximum load in variety PR107 was significantly (p < 0.05) lower than that in other varieties. The highest tensile strength was found in variety GT1 and the lowest in variety PR107. Both variety RRIM600 and variety Reken628 exhibited medium tensile strengths, but the significant (p < 0.05) differences in tensile strength were only observed between variety PR107 and variety GT1. Data analysis from Table 3 revealed that the elongation at break point (EBP) in variety GT1 was significantly (p < 0.05) higher than that in other varieties, with values of elongation at break point being 118.53%, 59.40%, and 52.20% greater than those in variety PR107, variety RRIM600, and variety Reken628, respectively. Figure 5 illustrates that bark samples from different varieties underwent deformation, and the strain in variety GT1 barks was greater than that in other varieties’ barks. According to Table 2, the Young’s modulus of variety RRIM600 barks was 13.25 MPa, 18.63 MPa, and 29.93 MPa greater than that of variety PR107, variety Reken628, and variety GT1 barks, respectively, but there were no significant (p < 0.05) differences among them.

3.6. Chemical Compositions Exhibited Diversities in Rubber Tree Barks of Different Varieties

The chemical compositions of the barks derived from the two-year-old twigs of different varieties of rubber trees are presented in

Table 4. Bark compositions of different varieties of rubber trees.

Variety	PC (%)	CC (%)	AILC (%)	HOC (%)
PR107	13.4000 ± 0.5859 b	1.8500 ± 0.0436 b	21.5000 ± 0.4359 a	58.9000 ± 0.5686 c
RRIM600	16.5000 ± 0.1732 a	0.8700 ± 0.0529 c	20.0000 ± 0.2646 b	60.1000 ± 0.0577 bc
Reken628	7.3000 ± 0.0700 c	3.1000 ± 0.0513 a	22.1000 ± 0.1528 a	69.6000 ± 0.5132 a
GT1	7.94000 ± 0.0208 c	0.3600 ± 0.0115 d	19.4000 ± 0.1155 b	60.4000 ± 0.3215 b

Data were represented as means ± SEs. Means that do not share the same letter(s) are significantly different at p < 0.05 by Duncan’s test of one-way ANOVA. PC, pectin content; CC, cellulose content; AILC, acid-insoluble lignin content; HOC, holocellulose content.

. The chemical compositions varied in the barks of four varieties of rubber trees. In terms of pectin content, it was found to follow a sequence of variety RRIM600 > variety PR107 > variety GT1 > variety Reken628, with respective pectin contents of 16.50%, 13.40%, 7.94%, and 7.30%. Notably, the pectin content in RRIM600 barks was significantly (p < 0.05) higher than that in the other varieties. When compared with those in three other varieties, the cellulose content in variety GT1 barks was the lowest (p < 0.05), being 81.62%, 60.92%, and 89.03% lower than that in PR107, RRIM600, and Reken628, respectively. Furthermore, these differences were statistically significant (p < 0.05). The value rank of acid-insoluble lignin content in barks was similar to that of cellulose content. Analysis of acid-insoluble lignin content exhibited that the barks in variety GT1 had the lowest value of it, and there were significant (p < 0.05) differences between variety GT1, variety PR107, and variety Reken628. Concerning holocellulose content, barks of variety Reken628 displayed the highest (p < 0.05) amount compared with barks in the other three varieties, but there were significant (p < 0.05) differences among variety PR107, variety Reken628, and variety GT1.

3.7. A Comprehensive Evaluation of Different Rubber Tree Varieties for Chilling Resistance

The fuzzy mathematical membership function method has been extensively used to evaluate the chilling resistance of plants, yielding satisfactory results. All physiological indicators were standardized for a comprehensive evaluation of chilling resistance using the chilling-resistance coefficient. The coefficients of each physiological indicator are presented in

Table 5. Chilling-resistance coefficients of varieties of different rubber trees.

Variety	Chilling-Resistance Coefficient										DM	Rank
	Chl a	Chl b	T Chl	Caro	Pn	Gs	Ci	Tr	Fv/Fm	ΦPSII
PR107	0.0615	0.0446	0.0092	0.0051	0.0388	0	0	0	0.0043	0.0101	0.1736	3
RRIM600	0.0788	0.0741	0.0124	0.0101	0.0071	0.0656	0.0402	0.0413	0.0059	0	0.3255	2
Reken628	0	0	0	0	0	0.0422	0.0052	0.0329	0	0.0071	0.0874	4
GT1	0.3278	0.3624	0.0539	0.0403	0.0231	0.0006	0.0127	0.0050	0.0027	0.0239	0.8524	1

. The sum of these coefficients was then used to calculate the membership function values for various rubber tree varieties, which served as the standard for comparison and assessment of chilling resistance (Table 5). Then, the membership function values of each indicator were ranked based on their numerical values in Table 5. The total membership function values of variety PR107, variety RRIM600, variety Reken628, and variety GT1 were 0.1736, 0.3255, 0.0874, and 0.8524, respectively. This elucidated that the chilling resistance of the four varieties increased in the order variety Reken628 < variety PR107 < variety RRIM600 < variety GT1. Thus, variety GT1 was found to have the strongest chilling resistance compared with the other three varieties.

We computed the entropy values and weights for ten indicators using Equations (5)–(8). As presented in

Table 6. Various weights of each indicator.

Indicator	Wvj	Ej	Wej	Wj
Chl a	0.3278	0.5887	0.1046	0.2109
Chl b	0.3624	0.5210	0.1218	0.2394
T chl	0.0539	0.5725	0.1087	0.0872
Caro	0.0403	0.5492	0.1146	0.0774
Pn	0.0388	0.6660	0.0849	0.0654
Gs	0.0656	0.5042	0.1261	0.1036
Ci	0.0402	0.5787	0.1071	0.0748
Tr	0.0413	0.6345	0.0929	0.0706
Fv/Fm	0.0059	0.7580	0.0615	0.0217
ΦPSII	0.0239	0.6949	0.0776	0.0490

Wvj, the weight of the j-th indicator by the membership function method; Ej, the information entropy of the j-th indicator; Wej, the entropy weight of the j-th indicator; Wj, combination weight.

, the entropy value of F_v/F_m is the biggest, so the weight of this indicator is the minimum, indicating that the difference in F_v/F_m among varieties is small. And vice versa. Based on Equations (8) and (9), we could ultimately derive the combination weights of various indicators and the comprehensive evaluation values of cold resistance for each variety. The rank is consistent with those calculated by the membership function method (

Table 7. Comprehensive evaluation values of chilling resistance in different varieties of rubber trees.

Variety	D	Rank
PR107	0.1957	3
RRIM600	0.4217	2
Reken628	0.1471	4
GT1	0.7457	1

D, the comprehensive evaluation value by the combination weighting method.

).

The comprehensive evaluation value was used as the chilling-resistance property in each variety, and it was integrated with latex burst data, bark physical properties, and their chemical components to construct a Spearman correlation matrix (Figure 6). This facilitated the determination of relationships among these variables. The results revealed that chilling resistance was strongly (p < 0.001) negatively correlated with both cellulose content and acid-insoluble lignin content, respectively. The total area of latex burst was significantly (p < 0.001) and positively correlated with holocellulose content and maximum load, respectively. The maximum load demonstrated a high (p < 0.001) positive correlation with holocellulose. Both displacement at the break point and elongation at the break point exhibited strong (p < 0.001) positive correlations with each other. Furthermore, there were strong (p < 0.001) correlations between cellulose and acid-insoluble lignin content.

4. Discussion

Global warming has resulted in increased climate instability and a higher frequency of extreme weather events, which have continuously negatively impacted agricultural productivity and crop yield over the past decades [23]. The escalating frequency of these extreme climates is causing a marked increase in severe cold damage to rubber trees, posing a significant challenge to natural rubber production. Global warming accelerates the phenological progression of rubber trees, thereby exposing them to sudden cold damage. Moreover, the risk of suffering cold damage has increased due to the northward acclimatization of the cultivation of rubber trees by global warming. Therefore, it is crucial to investigate the variations in chilling resistance and the impact of low temperatures on latex burst among different varieties of rubber trees. It is useful for the implementation of chilling-resistance cultivation measures and research on the mechanism.

4.1. Changes in Photosynthetic Pigment Content and Photosynthetic Capacity of Rubber Tree Seedlings in Response to Chilling Stress

Photosynthesis in plants is acutely sensitive to environmental temperatures and relies on factors such as chlorophyll pigment content, chlorophyll fluorescence, and photosynthetic parameters [24]. The chlorophyll pigment content is usually employed as an indicator of chloroplast development and photosynthetic capacity [25,26]. Chilling stress accelerates the decomposition of chlorophyll in leaves, impairs the synthesis substrate for chlorophyll, and consequently leads to a reduction in leaf chlorophyll pigment content [27]. In the current study, both chl a and T chl contents were found to be higher under 5 days of chilling stress conditions compared with those before the chilling stress. These findings align with our previous research and are consistent with observations in seedlings of other tree species [28]. It is possible that the alternations in endogenous hormone levels, signal transduction, and physiological response regulation in rubber tree seedlings are driven by the chilling stress [29]. Woody plants exhibit a certain degree of chilling resistance, which is influenced by prior chilling acclimation, and their genetic types play an important role in determining this resistance determination. Upon exposure to chilling stress, these plants activate their defense systems and increase chlorophyll contents to offset the reduction in chlorophyll contents caused by chloroplast injury [28]. Low-temperature stress results in the production of an excessive amount of reactive oxygen species (ROS), which have toxic effects on chlorophyll pigment contents [30]. Furthermore, ROS produced under low temperatures can specifically inhibit translation processes within the chloroplast [31]. Chlorophyll b (chl b) is particularly sensitive to chilling stress and is easily degraded [30]. Thus, the chl b content in rubber tree seedings experienced the most significant reduction after 5 days of chilling stress. When plants are subjected to multiple abiotic stresses, there has been a reported increase in carotenoid biosynthesis [32]. The influence of temperature on carotenoid metabolism in horticultural plants has been extensively researched [33,34]. Notably, variations in catenoid content across different tissues, development stages, or varieties can be significant at low temperatures [35]. For example, the accumulation of carotenoid content in specific banana cultivars [36] and the pulp of navel oranges [34] has been observed under conditions of low-temperature storage. Moreover, a treatment of sweet osmanthus petals at a low temperature (15 °C) resulted in an increase in carotenoid content [37]. Carotenoids have been associated with photoprotection against chlorophyll oxidation (ROS) in photosystems and light-harvesting complexes [38]. In an experiment involving avocado seedlings, carotenoid accumulation in leaves was greater under a −5 °C temperature treatment compared to those under 1 °C and −2 °C [39]. Similarly, the chilling stress significantly increased the carotenoid content in the leaves of rubber tree seedlings. These findings suggest that seedlings may be more sensitive to chilling stress than mature plants.

The parameters of chlorophyll fluorescence serve as indicators of light energy absorption and utilization in plant leaves. Therefore, the analysis of these parameters provides a useful technique for assessing the functionality of photosystem II (PSII) and chloroplasts [40,41]. Chilling stress suppresses the activity of PSII by diminishing photosynthetic efficiency [42]. In a study involving Arabidopsis leaves, those exposed to 4 °C treatment exhibited higher F_v/F_m values compared to those maintained at 22 °C/16 °C conditions after being placed in a cooling device, but there were no significant (p < 0.05) differences among them [43]. As our results in rubber tree seedlings showed, the reaction centers of PSII in leaves demonstrated some degree of chilling resistance during the initial stages of the chilling stress. As the duration of the stress increased, there was a decrease in photosynthetic efficiency and suppression of the photosynthetic electron transport rate of PSII, leading to damage to the reaction center of PSII. Ultimately, this resulted in an obstruction of PSII activity. In the current study, we observed a gradual decrease in both F_v/F_m and Φ_PSII in rubber tree seedlings subjected to chilling stress. The chilling-resistance variety exhibited less susceptibility to photoinhibition under low-temperature treatment. The maize variety Gurez, which is tolerant to low temperatures, has a lower photochemical efficiency of PSII (F_v/F_m) compared to the low-temperature-sensitive variety GM6. This typically leads to an increase in thermal energy dissipation [44], highlighting the inherent resistance of low-temperature-tolerant genotypes to sustained photoinhibition damage. In the experiment, the F_v/F_m value in variety GT1 was lower than that in variety PR107 and variety RRIM600 after 5 days of chilling stress, corroborating the aforementioned research perspectives. The magnitude of decrease in Φ_PSII in the cold-sensitive plant cultivar at low temperatures was greater than that in the cold-tolerant variety, indicating that the cold-tolerant variety experienced less photoinhibition under those conditions [45]. The reduction in light energy utilization efficiency at low temperatures is associated with an internal increase in stress stimulation, implying a general degradation of the photosynthetic electron transmission chain [46]. On the other hand, the damaged chloroplasts contribute to the decrease in the efficiency of photosynthetic electron transport and inhibition of PSII following exposure to chilling stress. Consequently, light energy is then converted into thermal energy, which the seedings dissipate through thermal energy dissipation [47]. During the period of chilling stress, the Φ_PSII values in variety GT1 remained high, indicating that the PSII of variety GT1 experienced less photoinhibition and that the light energy absorption of PSII photochemistry remained relatively stable under these conditions.

Low-temperature conditions exacerbate the imbalance between light energy absorption by the photosystem and plant metabolism, adversely affecting electron transduction, the Calvin cycle, and stomatal conduction. These conditions necessitate that plants adjust to reach a new balance for their growth and development [48]. Previous studies indicated that both stomatal and non-stomatal factors contribute to a reduction in photosynthetic rate. Stomata, as the conduits for carbon and water exchange between plants and their environment, provide an indication of plant metabolic function and are critical indexes of chilling stress [49]. The stomatal conductance (G_s) is not the sole limiting factor for photosynthetic rate. Other non-stomatal limiting factors that may constrain the photosynthetic rate include changes in photosynthetic pigment content, Rubisco content and activity, and photosynthetic assimilation [50]. In a one-day process of chilling stress, both net photosynthesis (P_n) and G_s in variety PR107 seedlings decreased, while intercellular CO₂ concentration (C_i) increased. This is attributed to non-stomatal factors impeding CO₂ utilization, leading to CO₂ accumulation. This included changes in photochemical activity, Rubisco content and activity, inorganic phosphate, and photosynthetic assimilation. In contrast, P_n in RRIM600 decreased, but C_i, G_s, and transpiration rate (T_r) in variety RRIM600 increased simultaneously. Here, the non-stomatal factors were identified as the primary restriction. As illustrated in Figure 1, alterations in the photosynthetic pigment content of RRIM600 may have enhanced these values. In Reken628 seedlings, both G_s and T_r exhibited consistent increasing trends, while P_n and C_i in them decreased concurrently. This suggests that stomatal conductance is not a primary limiting factor for P_n. It is likely that the activity of enzymes involved in photosynthesis was inhibited [51]. As depicted in Figure 3, P_n in GT1 seedlings was equivalent to G_s, C_i, and T_i. Stomatal closure resulted in insufficient CO_2, leading to photosynthetic inhibition in variety GT1 seedlings. During the 2–3 day chilling stress process, P_n, G_s, C_i, and T_r in variety PR107, variety RRIM600, and variety Reken628 seedlings decreased simultaneously. However, P_n in GT1 seedlings showed a slight recovery. To prevent excessive dehydration and chilling injury to leaves under low-temperature stress, plants typically reduce the degree of openness or ultimately close their stomata to avoid transpiration and water loss. The decline rate of G_s was more pronounced in the cold-sensitive cultivars than in the cold-resistant cultivars under aggravated chilling stress [51]. This phenomenon was also observed in the rubber tree seedlings. During a 4–5 day period of chilling stress, we noted an increase in P_n in both variety GT1 and variety RRIM600 seedlings. This rise can be attributed to the early exposure to chilling stress, which prompted the development of new coping strategies such as increased photosynthetic pigment content, heightened activity of enzymes involved in photosynthesis, and improved functionality of the chloroplast and photosystem to adapt to the new environment [51]. Afterwards, these seedlings gradually enhanced their ability to carry out photosynthesis under chilling stress. Furthermore, the decline in G_s in variety GT1 seedlings was less than that in other varieties compared with those before chilling stress. This could be due to the suppression of T_r in the leaves of variety GT1 rubber trees through metabolic regulatory pathways, which involve modulations in water transport channels, hormone levels, and signal transduction pathways [52]. In variety PR107 seedlings, P_n and G_s declined simultaneously, and C_i correspondingly decreased. Therefore, the decrease in P_n in variety PR107 seedlings may be due to the destruction of the balance between G_s and C_i [53]. The decline in P_n in variety Reken628 is likely attributed to impaired dark reactions [54], rather than stomata closure, as evidenced by the different dynamics of photosynthetic rate and stomatal conductance (Figure 3A,B).

4.2. Chilling Stress-Induced Bark Latex Burst and Nectar Secretion in Rubber Tree Seedlings

Rubber trees, subjected to various forms of cold damage, typically present symptoms indicative of harm. These include withered branches and leaves, black spots on the trunk, and latex burst across different sections of the tree’s body. Historically, mature rubber trees have frequently shown latex burst due to chilling stress. However, instances of latex burst in rubber tree seedlings are not commonly documented. Nonetheless, one-year-old seedlings have been observed to exhibit latex burst on their petioles and stems under conditions of chilling stress. In our prior research, we observed a significant negative correlation between the total number of latex burst positions in rubber tree seedlings and their chilling resistance [55]. Conversely, the total area of latex burst exhibited a weak correlation with the same trait. These findings are likely attributable to factors such as latex yield, dry rubber content, total solid content, the duration of latex flow, latex volume, and the number of laticifer rows present in various parts of the seedlings. In the current study, variety GT1 emerged as the most resistant variety. However, it did not exhibit the fewest number of latex-burst positions or the smallest total area of latex burst.

Although rubber trees possess both floral and extrafloral nectaries, the primary source of nectar secretion is from the extrafloral nectaries situated at the glandular points above the petiolule apex [10]. During honey production, bees exclusively gather nectar from these petiolule nectaries [2]. While all flowering rubber trees can secrete nectar from their petiolule nectaries, not all rubber trees with this secretion are flowering. This suggests that while flowering is an important indicator, it is not the sole determinant of petiolule nectar secretion. In our study, we observed nectar secretion in the nectaries of rubber tree seedlings under chilling stress, indicating an activation of their chilling-resistance mechanisms. Previous reports have noted that nectar secretion in mature rubber trees susceptible to wind or cold damage has been reported [56]. However, our observations revealed nectar secretion in one-year-old rubber tree seedlings. It is important to note that the ability to secrete nectar varies among different rubber tree varieties; hence, we did not observe visible nectar secretion in variety GT1 seedlings.

4.3. Bark Physical Properties, Chemical Components Were Correlated with Chilling Resistance and Latex Burst in Rubber Tree Seedlings

No singular indicator or mechanism can accurately assess the chilling resistance of plants, given that the variation patterns of identical indicators among different varieties of the same species are not entirely consistent. Utilizing a single physiological indicator to evaluate the cold resistance between varieties may result in a lack of objectivity. Meanwhile, the characteristic of cold resistance is complex and quantitative in rubber trees. Different varieties of rubber trees exposed to varying types of cold damage exhibit significant manifestations of harm [10]. The combination weighting method effectively circumvents the limitations of single weight calculation techniques, offering a more objective and comprehensive reflection of each indicator’s significance [22]. In this study, we employed this method to conduct a thorough evaluation of the cold resistance among four rubber tree varieties. The comprehensive evaluation value (D) served as the benchmark for a comparative analysis of the chilling resistance; a higher D value indicated greater resistance to chilling stress in the respective rubber tree variety. Our findings revealed that variety GT1 exhibited the most robust chilling resistance, while variety Reken628 demonstrated the weakest.

The mechanical properties of barks are primarily determined by their chemical compositions [57]. The amalgamation of individual fibers into fiber pairs markedly augments their biophysical properties, including tensile strength and Young’s modulus. When this enhanced biophysical behavior is extended to the entire bark structure, it most likely confers a beneficial response during and following mechanical action. Key determinants of bark tensile properties include the high cell wall content of the bark fibers, the method of connection between adjacent fibers, and the alignment of fibers within interwoven layers [58]. It is noteworthy that the composition of the cell wall may vary across organs, tissues, species, and even different developmental stages. Cellulose, an abundant linear homopolysaccharide, forms the directional organization of rigid cellulose microfibrils and serves as the main-bearing structure [59]. An increase in bark thickness amplifies the maximum tensile load of the bark [57]. As demonstrated in Table 3, the bark thickness in variety Reken628 was significantly greater than that in other varieties, with the maximum load in this variety being the highest. The tensile strength improved as fiber loading increased. The enhanced tensile properties of the barks are responsible for their increased tensile strength. However, an increase in cellulose content may lead to poor adhesion, potentially resulting in a reduction in tensile properties [60]. Therefore, the cellulose content of variety GT1 bark was found to be the lowest among all varieties, yet it exhibited the highest tensile strength. This observation aligns with Bjurhager’s report that a reduction in lignin content results in a slight but significant decrease in tensile stiffness [61]. As demonstrated in Table 4, the acid-insoluble lignin content and Young’s modulus value were both lowest in variety GT1 bark (Table 3). Pectin, particularly abundant in the middle lamella, plays an essential role in regulating wall properties and adjusting wall extensibility by affecting the alignment of cellulose microfibrils [62]. In the context of wood, initial deformation under constant loading is elastic. However, as the duration of this constant-loading increases, the nonelastic components begin to respond, leading to a decrease in the elastic component [58]. When barks are subjected to a critical strain level, the fibers may lose contact due to relatively weak intercellular adhesion, resulting in the disruption of cell-to-cell bonding and tissue failure [63]. On the other side, the presence of more flaws, barks can trigger rapid failure in tensile tests [64]. Among the rubber tree varieties studied, variety GT1 barks exhibited the highest elongation, indicating superior flexibility, but also the weakest rigidity, as evidenced by the stress–strain curve in Figure 5.

5. Conclusions

In summary, our results showed the chilling stress caused significant differences in latex burst and nectar secretion among one-year-old rubber tree seedlings. Chilling resistance and latex burst status were positively correlated with the chemical compositions of two-year-old twig barks corresponding to each variety. Moreover, the latex burst status was negatively correlated with the tensile property of the barks, as determined by a multivariate analysis of the experimental data. These findings suggested that chemical compositions and biophysical properties play roles in chilling resistance and latex-burst characteristics. Therefore, these factors warrant attention in future endeavors. Nonetheless, additional investigations are necessary to elucidate the roles and mechanisms of nectar secretion in the latex-burst characteristics of rubber trees subjected to chilling-stress conditions. Furthermore, it would be interesting to broaden this study to encompass the physiological metabolism, biochemical, and molecular mechanisms underlying latex burst in rubber trees under chilling stress.