Comprehensive evaluation of physiological response and cold tolerance of domesticated Cinnamomum camphora (L.) Presl under low temperature stress

Material selection and low temperature treatment of Cinnamomum camphora (L.) Presl

Cinnamomum camphora (L.) Presl trees were chosen from a garden green space in Shangqiu city, Henan Province, China. Shagnqiu city is located between 114°49′-116°39′ N, 33°43′-34°52′E, at an altitude of 50 m. The cultivation years (y.) of the chosen plants were 1 y, 5 y, 10 y, and 15 y. The best period of the experiment needs is April-May or August-October. Ten plants of each cultivation period with the same growth potential were planted in the same direction and position of the crown, and annual branches with a diameter of 0.20−0.25 cm were chosen, with 10 leaves in the same position of each branch, and three repetitions in each treatment. All sampling was done while being cautious to avoid hurting branches in the process of picking the leaves. The collected Cinnamomum camphora (L.) Presl leaves are cleaned, dried, and put into self-sealing bags for later use. Each repeated leaf was divided into six parts on average, one part was stored in a refrigerator at 4 °C as the control, and the other five parts were treated at 0 °C, −5 °C, −10 °C, −15 °C, and −20 °C, respectively, and then dropped to the target temperature at a rate of 4 °C/h for 24 h. They were then gradually heated to 4 °C in the same way and placed for 12 h, and then the cold tolerance related indexes were measured.

Physiological and biochemical parameters

The determination method is based on the principles and techniques outlined in the textbook “Physiological and Biochemical Experimental Principles and Technology”, authored by He (2000).

Comprehensive evaluation method of cold tolerance

The cold tolerance of Cinnamomum camphora (L.) Presl was comprehensively evaluated by principal component analysis and the weighted membership function method (Ying et al., 2023), and the calculation methods were as follows:

(1)${\displaystyle U\left(Xi\right)=\frac{\left(Xi-Xmin\right)}{\left(Xmax-Xmin\right)}\left(i=1,2,3,4\dots n\right)\left(\text{positive correlation}\right)}$ (2)${\displaystyle U\left(Xi\right)=1-\frac{\left(Xi-Xmin\right)}{\left(Xmax-Xmin\right)}\left(i=1,2,3,4\dots n\right)\left(\text{negative correlation}\right).}$

In Eq. (1): U (Xi) is the membership function value of index i; Xi is the measured value of the index i; X_max and X_min are the minimum and maximum values of the index i, respectively.

(3)${\displaystyle Wi=Pi/\sum _i^{n}Pi\left(i=1,2,3,4\dots n\right)}$ (4)${\displaystyle D=\sum _{i=1}^n\left[U\left(Xi\right)\times Wi\right]\left(i=1,2,3,4\dots n\right).}$

In Eqs. (3) and (4), Pi represents the contribution rate of the i comprehensive index in the principal component analysis, Wi represents the weight, and D represents the evaluation value of the comprehensive index for different cultivation years.

Data processing and analysis

Data processing and graphing were performed using Microsoft Office Excel 2010, while variance analysis (ANOVA), correlation analysis, and principal component analysis (PCA) to assess Cinnamomum camphora (L.) Presl cold tolerance were conducted using SPSS 26.0 software (IBM, Armonk, NY, USA).

Effects of low temperature stress on cell membrane damage of Cinnamomum camphora (L.) Presl leaves with different cultivation lengths

The relative electrical conductivity, relative water content, and MDA content of plant leaves under low temperature stress reflects the damage degree of the plant cell membrane, and the lower the value, the less affected it is by stress. Figures 1–3 show that with the decrease in temperature, the change trend of relative electrical conductivity (Fig. 1) and MDA content (Fig. 2) of Cinnamomum camphora (L.) Presl leaves increased gradually as the temperature decreased. The relative water content (Fig. 3) decreased as the temperature decreased, but the change ranges differed.

The relative electrical conductivity change ranges of Cinnamomum camphora (L.) Presl leaves at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 26.26%–13.98% , 30.71%–19.24%, 37.36%–27.18%, 44.16%–32.24%, 63.21%–52.05%, and 86.43%–76.24%. respectively (Fig. 1). The results of a matched sample T-test showed that the relative electrical conductivity of Cinnamomum camphora (L.) Presl leaves varied significantly (P <0.05; Table 1).

The MDA content change ranges of Cinnamomum camphora (L.) Presl leaves at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 12.10 mmol/g−3.25 mmol/g, 15.90 mmol/g−4.08 mmol/g, 10.53 mmol/g−2.05 mmol/g, 23.20 mmol/g−5.35 mmol/g, 31.30 mmol/g−5.89 mmol/g, and 36.47 mmol/g−8.13 mmol/g, respectively (Fig. 2). The single factor analysis of variance showed that the MDA content of Cinnamomum camphora (L.) Presl leaves varied significantly between different lengths of cultivation (P <0.05; Table 1)

The relative water content change ranges of Cinnamomum camphora (L.) Presl leaves at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 95.35%-65.92%, 71.36%-49.67%, 54.67%-34.89%, 43.12%-23.12%, 26.03%-11.21%, and 23.03%–8.15%. respectively. The single factor analysis of variance showed that the relative water content of Cinnamomum camphora (L.) Presl leaves varied significantly between different lengths of cultivation (P <0.05; Table 1). The relative comprehensive analysis of Cinnamomum camphora (L.) Presl leaves from trees with different lengths of cultivation showed that as cultivation time increased, the adaptability of Cinnamomum camphora (L.) Presl was enhanced, and low temperature damage in the cell membrane of Cinnamomum camphora (L.) Presl leaves was weakened (Fig. 3).

Effects of low temperature stress on osmoprotectants in Cinnamomum camphora (L.) Presl leaves with different cultivation lengths

Osmoprotectants provide a regulation mode under low temperature stress. The osmotic potential of cell fluid is reduced by actively accumulating lytic substances in cells. In order to prevent excessive cell water loss under low temperature stress, the change trend content of soluble protein (Fig. 4) in Cinnamomum camphora (L.) Presl gradually increased, did not differ by length of cultivation. The content of soluble sugar (Fig. 5) and free proline (Fig. 6) increased in an S-shaped curve by cultivation year.

It can be seen from Fig. 4 that the soluble protein content change ranges of Cinnamomum camphora (L.) Presl leaves at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 3.51−6.18 mg/g, 6.24−9.95 mg/g, 9.44–19.59 mg/g, 14.23–28.36 mg/g, 17.34–33.19 mg/g, and 25.15–32.23 mg/g respectively. Therefore, the soluble protein content of Cinnamomum camphora (L.) Presl leaves increased gradually with the increase of cultivation years, and the difference was significant (P <0.05; Table 1). The maximum value of soluble protein appeared at −15 °C in trees cultivated for 10 y or 15 y and at −20 °C in trees cultivated for 1 y or 5 y.

Changes in soluble sugar content in Cinnamomum camphora (L.) Presl leaves under low temperature stress are shown in Fig. 5. As the temperature decreased, the soluble sugar content gradually increased. From 1 y to 15 y, the soluble sugar content change ranges at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 9.64–26.97 mg/g, 15.37–39.86 mg/g, 26.63–53.97 mg/g, 45.49–76.75 mg/g, 52.74–81.24 mg/g, and 55.61–85.34 mg/g respectively. As cultivation time increased, the soluble sugar under low temperature stress also increased, and the difference was significant (P <0.05; Table 1).

The changes of free proline content in Cinnamomum camphora (L.) Presl leaves under low temperature stress are shown in Fig. 6. The free proline content gradually increased as the temperature decreased. From 1 y. to 15 y, the free proline content change ranged at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 55.83–85.23 µg/g, 68.95–89.87 µg/g, 95.38–214.38 µg/g, 219.19–389.89 µg/g, 321.28–453.65 µg/g, and 381.23–478.96 µg/g, respectively. As years of cultivation increased, the free proline under low temperature stress also increased and the difference was significant (P <0.05; Table 1); for free proline, the change range was the largest at −10 °C, with 170.10 µg/g cultivated for 1 to 15 years, which increased by 77.88% compared with 1 y.

Effects of low temperature stress on protective enzymes of different cultivation lengths Cinnamomum camphora (L.) Presl leaves

Protective enzymes help regulate a plant under low temperature stress, as endogenous active oxygen species scavengers, CAT, SOD and POD can remove excess active oxygen species in the body to a certain extent, maintain the metabolic balance of active oxygen species, and protect the membrane structure, thus reducing the damage of active oxygen species in plants, so that Cinnamomum camphora (L.) Presl has the ability to tolerate low temperature stress. It can be seen from Figures 7–9 that the change trends of CAT activity (Fig. 7), SOD activity (Fig. 8), and POD activity (Fig. 9) of Cinnamomum camphora (L.) Presl leaves under different cultivation years under low temperature stress are basically consistent.

Under low temperature stress, the change trend of CAT activity in Cinnamomum camphora (L.) Presl leaves did not differ by length of cultivation (Fig. 7), with CAT activity first decreasing, then increasing, and then decreasing again as the temperature decreased. The CAT activity of Cinnamomum camphora (L.) Presl decreased from 4 °C to 0 °C, and increased significantly from 0 °C to −15 °C. When the temperature was lower than −15 °C, the CAT activity decreased sharply, indicating that the CAT activity reached a maximum at about −15 °C. Cultivation for 15 years is better than 1 year, the increments of CAT activity at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 31.72%, 58.99%, 49.87%, 53.88%, 40.21% and 52.27%, respectively.

Under low temperature stress, SOD activity of Cinnamomum camphora (L.) Presl leaves first increased and then decreased as the temperature decreased (Fig. 8), with SOD activity reaching its highest point at −5 °C. Cultivation for 15 years is better than 1 year, the increments of SOD activity at 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C were 152.60%, 218.33%, 331.07%, 192.81% and 316.11% respectively. The analysis of variance showed that when the temperature was above −15 °C, as the temperature decreased, the stimulating effect of cultivation years on SOD activity of Cinnamomum camphora (L.) Presl was gradually enhanced.

The response trend of low temperature stress to POD activity in Cinnamomum camphora (L.) Presl was consistent by length of cultivation (Fig. 9). When the temperature decreased from 4 °C to 0 °C, the POD activity of Cinnamomum camphora (L.) Presl leaves decreased, When the temperature decreased from 0 °C to −10 °C, the POD activity of Cinnamomum camphora (L.) Presl leaves increased significantly. When the temperature was lower than −10 °C, the POD activity of Cinnamomum camphora (L.) Presl leaves decreased sharply after reaching a maximum around −10 °C.

Under the temperature treatment of 4 °C, 0 °C, −5 °C, −10 °C, −15 °C and −20 °C, the POD activity of Cinnamomum camphora (L.) Presl leaves cultivated for 1 to 15 years changed by 38.98%, 62.74%, 27.45%, 70.93%, 73.88%, and 26.70%, respectively, indicating that the POD activity of Cinnamomum camphora (L.) Presl leaves gradually increased as the temperature decreased until −15 °C.

Correlation analysis and matched sample T-test of different cultivation years on cold tolerance index of Cinnamomum camphora (L.) Presl

Table 1 shows that longer cultivation time of Cinnamomum camphora (L.) Presl is positively correlated with the physiological index of Cinnamomum camphora (L.) Presl, and the difference is significant, except for SOD. The difference in SOD changes between 1 y and 5 y of cultivation is not significant, but reaches significance after 5 y of cultivation.

Comprehensive cold tolerance and principal component analysis of cold tolerance e of the Cinnamomum camphora (L.) Presl in different cultivation years

The principal component analysis of nine physiological indexes of Cinnamomum camphora (L.) Presl leaves related to cold tolerance showed that the contribution rate of the first two principal components were 52.01% and 32.89%, and the cumulative variance contribution rate was 84.90% (Table 2).

The first principal component corresponds to the larger eigenvectors of soluble protein content, soluble sugar content, and free proline content, which reflect the change of osmoprotectants content, and the relative water content reflects the membrane permeability. The second principal component corresponds to the larger eigenvector of SOD enzyme activity, which reflects the change of the protective enzyme system.

Using Eq. (3), the weights of the two comprehensive indexes were 0.61 and 0.39, respectively. The weighted membership function value D was then calculated under each treatment using Eq. (4), and sorted according to the D value (Table 3). Table 3 shows that more cultivation years improves the cold tolerance of Cinnamomum camphora (L.) Presl.

Conclusion

In this experiment, the membership function method was used to comprehensively evaluate the cold tolerance of the Cinnamomum camphora (L.) Presl in four separate cultivation years. The cold tolerance of trees that had been cultivated for 15 y was the strongest, and was the weakest in those that had only been cultivated for 1 y, which indicates that the cold tolerance of the Cinnamomum camphora (L.) Presl can be improved by lengthening cultivation time. For planting these trees in the north, domestication cultivation and moving cultivated trees from the south to the north area may provide better outcomes. In the initial transplanting stage of Cinnamomum camphora (L.) Presl, anti-freezing protection measures should be taken to avoid huge economic losses. A principal component analysis showed that relative electrical conductivity, soluble protein, soluble sugar content, and catalase activity could be used as important indexes for cold tolerance analysis of Cinnamomum camphora (L.) Presl. and the cumulative variance contribution rate was 84.90%. Among these factors, relative electrical conductivity was the most representative. These results could provide some reference for seed selection or northward migration of Cinnamomum camphora (L.) Presl.

The important physiological parameters changed sharply at minus 10–15 degrees. Thus we extrapolated that the critical temperature of the cold damage of Cinnamomum camphora (L.) Presl was in the range of minus 10–15. Which is consistent with the research results Jin et al. (2023).

The results of this study not only provide theoretical support for screening cold tolerance varieties of Cinnamomum camphora (L.) Presl, but also provide a basis for further study of the physiological mechanism of the Cinnamomum camphora (L.) Presl under low temperature stress. However, molecular differences of cold-tolerance varieties of Cinnamomum camphora (L.) Presl with different cultivation years need to be further explored.