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［Objectives］ This study aimed to select evergreen broad-leaved woody plants with higher ornamental value and stronger cold tolerance for introduction from the south to the north, and to apply them to urban greening, so as to enrich the plant community structure of the landscape in the northern region.
［Methods］Three species of evergreen broad-leaved woody plants, i.e., Ligustrum lucidum, Ilex cornuta and Eriobotrya japonica, were selected as the experimental materials. The morphological performances and the changes of the physiological indexes were observed and measured during the overwintering period in the open field in Beijing. The relationship between the indexes and the low temperature was also analyzed. The strength of cold tolerance of the three species was compared.
［Results］ The electrical conductivity, the contents of MDA and proline were negatively correlated with the corresponding low temperature. The contents of soluble sugar and soluble protein increased with the dropping temperature, but they had little response to the short-term temperature rise.
［Conclusions］Combined with morphological and physiological indexes, it was found that the changes of the contents of proline and soluble sugar among the physiological indexes were closely related to the cold tolerances of the three tree species of broad-leaved woody plants. The cold tolerance of I. cornuta was the strongest, E. japonica was the second, and that of L. lucidum was the worst.
Key words Evergreen broad-leaved woody plant; Cold tolerance; Overwintering
Received: January 12, 2021 Accepted: March 6, 2021
Supported by Key Project of Beijing Municipal Education Commission; Project of Construction of Advanced Horticulture Under Beijing Municipality (2020); National Natural Science Foundation of China (31201645; 31640070); Beijing Municipal Natural Science Foundation (3172006); Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT 20150503).
Sifan LI (1997-), female, P. R. China, Master, devoted to research about cultivation physiology of ornamental plants.
*Corresponding author. E-mail: shenman@vip.sina.com.
Beijing is located in the subhumid area of the warm temperate zone, and has a typical continental monsoon climate. Its natural zonal plant communities are mixed coniferous and deciduous broad-leaved mixed forests. The woody plants in such natural communities consist of a large number of deciduous trees and shrubs and a small number of evergreen coniferous trees. The composition of such natural communities determines that conifers and deciduous broad-leaved trees are the main landscaping tree species in Beijing, and evergreen broad-leaved trees are rarely used. Therefore, compared with the three seasons of spring, summer and autumn, Beijings winter greening landscape is monotonous and lacks vitality［1］. If some southern evergreen broad-leaved trees with high ornamental value and able to withstand the low temperature in winter in the north can be introduced to Beijing, they will not only enrich the tree species diversity in the urban garden landscape, but also optimize the landscape community structure of the garden plants in the northern region and improve the ecological environment of the northern region. The urban landscaping department has been promoting the northward migration of evergreen broad-leaved plants. However, based on the current introduction, the types of evergreen broad-leaved tree species in Beijing are still relatively few and it is difficult to promote new varieties. The main reason is the geographical environment, especially the low temperature in winter, which severely restricts the normal growth of evergreen broad-leaved trees in northern regions［2］.
The climate of Beijing is characterized by dry and cold winter with low rainfall, low humidity, large temperature difference between day and night and strong wind, and dry and windy spring with a temperature rising rapidly. This condition will cause repeated freezing and thawing of plant leaves and thus damage. Many evergreen tree species that grow at latitudes higher than Beijing and lower temperatures than Beijing are difficult to be cultivated in Beijing［3-5］. In the work of introducing evergreen broad-leaved plants, it was found that due to the large annual changes in natural climate conditions, short-term introduction success is not enough to support the introduction of tree species to adapt to local natural conditions, and the inadaptability of some tree species to low temperature cannot be observed in a short period of time. Therefore, it is very necessary to observe the evergreen broad-leaved plants introduced in northern regions for a long term and study their low temperature tolerances.
Based on the above research background, in this study, three common evergreen broad-leaved trees in East China were introduced into Beijing to observe the differences in plant leaf morphology during the natural overwintering process, analyze the changes in physiological indexes related to cold tolerance and their correlation with natural cooling, and compare the strength of cold tolerance of the three tree species, so as to provide experimental reference basis for establishing evaluation indexes of cold tolerance of evergreen broad-leaved plants and selecting cold-tolerant evergreen broad-leaved tree species suitable for planting in Beijing area.
Materials and Methods
Experimental materials
The plant materials are Ligustrum compactum, Ilex cornuta, and Eriobotrya japonica seedlings with a larger soil balls introduced from Yanling County, Xuchang City, Henan Province (belonging to the northern subtropical evergreen broad-leaved plant distribution area) in April 2002. They were planted in the office area and family residential area of the Fangshan Highway Branch in Liangxiang West Road of Fangshan District, Beijing. The plants were located in the planting area with good microclimate conditions (leeward and sunny, 2 to 4 m away from the buildings on the south and west sides of the buildings). At the early stage of introduction (2002-2006), winter was supplemented with cold-proof measures, that is, blue plastic film cold-proof sheds were used, grass ropes were wrapped around the trunk and main branches of the plants, and the base of the trunk was cultivated. Conventional water and fertilizer cultivation management was adopted. Experimental methods
Leaf shape observation
From November 2015 to April 2016, L. compactum, I. cornuta, and E. japonica at the Fangshan test site were observed and photographed every 2 weeks, and the morphological indexes of plant leaves were recorded. The daily weather conditions were recorded according to the weather forecast. The daily average temperature and daily minimum temperature in a small environment were measured using a GGL-20 temperature and humidity automatic recorder, and the monthly average maximum temperature and monthly average minimum temperature were calculated.
Determination of physiological indexes
Mature leaf samples were collected at 10:00 am throughout the winter (November 2015 to March 2016) for the determination of physiological and biochemical indexes. L. compactum and E. japonica leaves could not be sampled from February to March 2016 due to browning and withering. The specific sampling time were as follows: ① early winter, the stable temperature period: November 19, 2015, ② the sudden temperature drop period: November 28, 2015 (on November 22, there was a heavy snowfall in Beijing area, and the temperature dropped sharply, so the sampling was increased once in November, and noted in the figure as "11t"), ③ the period of gradual decrease in temperature: from mid-December 2015 to mid-January 2016, and ④ the period of warming in early spring: mid-February to mid-March 2016.
The various physiological indexes were carried out according to the method of Li［6］. The relative conductivity was determined by the conductivity method; the malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) method; the proline content was determined by acid ninhydrin method; the anthrone colorimetric method was used for the determination of soluble sugar content; and the Coomassie brilliant blue method was used for the determination of soluble protein content.
Statistics and analysis
The experimental data were analyzed by variance analysis and graphed using Microsoft Excel, GraphPad Prism 7.0 and SPSS 17.0 software.
Results and Analysis
Changes in leaf morphological indexes of three evergreen broad-leaved trees during overwintering
Plant leaves showed the most obvious changes during overwintering. From October 2015 to April 2016, the changes in leaf color and leaf shape of these three evergreen broad-leaved plants were observed and recorded (Fig. 1). As the winter temperature dropped, the leaves of the three tree species were brownish and slightly curled and turned. At the extreme low temperature on January 3, 2016, the leaves of the three tree species deeply browned, suffered from more serious water loss and even fell off. In the deep winter of February, the leaves of L. compactum were all browned, dried, and easy to fall off. By March, the leaves on the annual branches fell off and the branches became bald. From December, part of the leaves of I. cornuta were browned but the plants were normal as a whole. By March, the leaves were partially curled and dried, and the fruit was all browned, and more likely to drop. For E. japonica, only a small amount of leaves changed their color in January, the plants were normal as a whole, and the leaf color still changed less until March. As the temperature rose in early spring, the branches of L. compactum, I. cornuta and E. japonica grew out new leaves one after another in April.
Combined with the observation results of leaf texture, it was found that the leaves of L. compactum were thin, the leaves of I. cornuta were hard and leathery, and the leaves of E. japonica were covered with hairs. We believes that during the overwintering period in the open field, as the temperature drops, the leaves of L. compactum suffer from frost damage because of their thin texture, while the leaves of E. japonica are protected by hairs, can relatively reduce water loss and suffer less frost damage, and because of the wax on the leaf surface, I. cornuta is least affected by low temperature. From the perspective of leaf morphology, the order of cold tolerance of the three evergreen broad-leaved tree species was: I. cornuta>E. japonica>L. compactum.
Changes in physiological indexes of three evergreen broad-leaved plants during overwintering
Changes in relative conductivity and malondialdehyde content of leaves during overwintering
The different letters mean significant differences (P<0.05). The same below.
Sifan LI et al. The Comparison of Cold tolerance of Three Species of Evergreen Broad-leaved Woody Plants
Cell membrane is the primary part of plants that is damaged by low temperature［7］. The conductivity of the leaves can reflect the degree of chilling damage to the leaves of plants. The relative conductivity of plants is usually determined to reflect the degree of freezing damage to plants. The principle is that when plants are severely frost-damaged, a series of dynamic changes will occur in the cell membrane, leading to the exosmosis of electrolytes in plant cells［8］. It can be seen from Fig. 2 that as the temperature decreased, the conductivity of the leaves of the three evergreen tree species increased, and when the temperature rose, the electrical conductivity decreased again, which was consistent with the law of temperature changes. Specifically, at the sudden temperature drop on November 28, 2015, the conductivity values of the leaves of the three tree species all rose rapidly, and were significantly higher than those in other time periods. By December, the conductivity of the three tree species adapted to the continuous low temperature decreased again to the original level. During the whole overwintering process, the conductivity of I. cornuta and L. compactum changed significantly between different months, indicating that the conductivity changes were closely related to low temperature, while the value of E. japonica did not change significantly.
MDA content is the product of membrane lipid peroxidation, and the change of its content reflects the degree of damage to plants, and is one of the important signs of plant membrane damage by adversity［9］. Therefore, determining the MDA content is an effective method to determine the cold resistance of plants.
Fig. 2 shows that the MDA contents of the three broad-leaved plants all increased with the decrease of temperature, and all reached their maximums in December 2015. After a sharp drop in temperature on November 22, the MDA content of I. cornuta rose significantly, reached the highest value in December, and then slowly decreased, indicating I. cornutas response to the sudden drop in temperature. The change trends of MDA content in L. compactum and E. japonica were similar to that of I. cornuta. They reached the highest value in December and then decreased, but the increase rates of the two were higher than that of I. cornuta. The decreases of the MDA contents in the leaves of L. compactum and E. japonica in January might be due to the browning and drying of the leaves. In November, the MDA content of I. cornuta was significantly lower than those of L. compactum and E. japonica, indicating that I. cornuta had better cold tolerance than the former two.
Changes in proline and malondialdehyde contents of leaves during overwintering
The decrease in water content of plant tissues after being subjected to low temperature stress is conducive to the accumulation of dry matter in the body, which leads to the increase of cold resistance of plants［10］. Fig. 3 shows that during the natural temperature declining process, the leaf water contents of the three broad-leaved plants showed an overall downward trend. Among them, the water content of E. japonica dropped rapidly when the temperature suddenly dropped on November 22, and then showed an upward trend in December. I. cornuta and L. compactum showed a continuous downward trend during the entire temperature declining process, and the water content of I. cornuta rose when the temperature rose in March. The leaf water contents of the three tree species were negatively correlated with temperature changes. Proline is an important protective substance for cold resistance. Because of its strong hydrophilicity, it can stabilize the metabolic processes in protoplast colloids and tissues, thereby reducing the freezing point of cells, so its content is related to the cold resistance of plants［11］. Fig. 3 shows that the proline contents of the three evergreen broad-leaved tree species all increased with the decrease of temperature, and when the temperature rose, the proline content of I. cornuta decreased again. Except for the highest proline content of E. japonica, which appeared in December, the highest proline contents of L. compactum and I. cornuta appeared in January 2016. During the sudden temperature drop in November 2015, the proline contents of E. japonica and L. compactum showed no significant changes.
From the perspective of the overall change trend, the change values of proline content were arranged from large to small as: I. cornuta﹥E. japonica﹥L. compactum, and the differences were significant.
Changes in soluble sugar and soluble protein contents of leaves during overwintering
Soluble sugars are important metabolites in plants. After being stressed, plants will actively accumulate some soluble sugars to reduce osmotic potential and freezing point, so as to adapt to changes in external environmental conditions［12］.
Fig. 4 shows that the soluble sugar contents of these three evergreen broad-leaved plants generally increased gradually as the temperature decreased. Due to the sudden temperature drop on November 22, the soluble sugar contents of the leaves of the three broad-leaved tree species increased sharply between 11t and December. L. compactum reached its maximum value in December, and afterwards, a large number of leaves were browned due to severe freezing damage, so the soluble sugar content of leaves decreased in January. The soluble sugar content of E. japonica leaves reached a maximum in January 2016. The soluble sugar content of I. cornuta showed an overall upward trend, and reached its maximum in February. And compared with other two tree species, the soluble sugar content of I. cornuta was relatively high, so it was inferred that I. cornuta had stronger cold tolerance.
Regarding the relationship between soluble protein and plant cold resistance, some researchers believe that soluble protein is highly hydrophilic, and can increase the water holding capacity of cells and reduce damage caused by icing, thereby improving the cold resistance of plants［13］. It was found from this study that the soluble protein contents of the three evergreen broad-leaved plants showed an upward trend as the temperature dropped. The soluble protein contents of L. compactum and E. japonica reached their maximums in December during the low temperature period from the temperature decrease in November 22 to December (Fig. 4). From the overall change trend, the maximum soluble protein contents of I. cornuta, E. japonica and L. compactum all appeared in January 2016, and they were negatively correlated with sub-zero low temperature in the deep winter (from later November to January). During the whole winter temperature declining process, the differences between the various tree species changed significantly, and the soluble protein content changes ranked as L. compactum﹥I. cornuta﹥E. japonica. Conclusions and Discussion
During the overwintering period of plants in the open field, in addition to measuring the relevant physiological indexes of plants to judge their cold tolerance (resistance), it is also necessary to observe the growth status and morphological performance of the plants during the overwintering process. Wan et al.［5］ observed the overwintering performance of evergreen broad-leaved plants such as Buxus megistophyll, B. sinica, Ligustrum lucidum, Pyracantha fortuneana, Yucca gloriosa, Photinia serratifolia. and Hedera nepalensis var. sinensis by the above method and identified the cold resistance of these plants. In this study, we also observed the overwintering performance of 3 evergreen tree species introduced in Beijing and cultivated in Fangshan area. According to the morphological changes of each tree species, it was found that the different texture and structure of the leaves would affect their cold resistances, and during the winter, L. compactum turned from evergreen to semi-deciduous through leaves to adapt to the low temperature and drought in winter in Beijing.
During the overwintering period, the changes in the external morphology of plants can only show the response of plants to low temperature to a certain extent, and cannot quantify the cold tolerances of plants. Therefore, it must be combined with the measurement of physiological indexes to make a comprehensive judgment. Plant cells communicate with the natural environment through their cell membranes, and the impact of low temperature stress on plants first acts on the plasma membrane. When plants are injured by low temperature, the unsaturated fatty acids in the membrane will undergo membrane lipid peroxidation, and the final product MDA accumulated will damage the cell membrane and cause electrolyte leakage. Therefore, measuring the conductivity value and MDA content of plants can reflect the degree of damage to the cell membrane and the degree of cold resistance of plants［14-15］. After plants are injured by low temperature, the lower the relative conductivity and MDA content, the stronger the cold tolerance of plants; otherwise, the weaker the cold tolerance［16］. In this study, it was found that the conductivity of L. compactum during the overwintering period was the highest, and its cold resistance was the worst; and the MDA contents of L. compactum and E. japonica leaves were higher than that of I. cornuta, indicating that the cold resistance of I. cornuta was stronger than that of L. compactum and E. japonica. Proline is an important cold-resistant protective substance in plants. When the temperature decreases, its content increases rapidly, indicating that plants cold resistance is enhanced. When the adversity is relieved, the content of proline decreases rapidly［17］. Jiang et al.［18］ compared the cold resistances of different strains of Citrus medica and found that the proline contents of the strains with strong cold resistance maintained a higher level. Therefore, the strength of cold resistance of plants can be judged by measuring the content of proline. In this study, the proline content of the leaves of the three tree species all increased with the decrease of temperature. The changes of the proline content ranked as I. cornuta﹥E. japonica﹥L. compactum, indicating I. cornuta had the strongest cold resistance, followed by E. japonica and L. compactum was the weakest, which was consistent with the results obtained by morphological observation.
As cell contents, soluble sugar and soluble protein are also important protective substances for cold resistance of plants, which can maintain the structural stability of plant cells, regulate osmotic pressure, and play a positive role in resisting cold damage to plants［19］. Under low temperature stress, plants have been proved by a large number of experiments by increasing the content of soluble sugar in the body to improve cold resistance. Therefore, the contents of soluble sugar and soluble protein can be used to identify the cold resistance of plants［20-23］. It is generally believed that the content of soluble protein increases with the increase in cold resistance under low temperature stress, but there are also reports pointing that the increase in the content of soluble protein in some plants has no correlation with their cold resistance［24］. In this study, the soluble sugar content of I. cornuta was higher than that of E. japonica and L. compactum, indicating that the cold tolerance of I. cornuta was higher than that of E. japonica and L. compactum. From the perspective of changes of soluble protein contents in the leaves of the three tree species, L. compactum had the largest change, followed by I. cornuta, and E. japonica had the smallest change.
To sum up, during the whole wintering period of the three evergreen broad-leaved trees, there was certain correlation between the measurement results of various physiological indexes and the natural low temperature and the cold tolerances of the tree species. However, the cold tolerance of evergreen broad-leaved plants cannot be measured by a single physiological index, but instead, a comprehensive analysis should be carried out among various physiological indexes, combined with plant morphological indexes, tree species characteristics and complex environmental factors. In the analysis of various indexes in this study, it was found that the proline content and soluble sugar content were closely related to the cold tolerances of the three evergreen broad-leaved trees and also consistent with the observation results of the morphological changes of the plants during overwintering, and thus can be screened as indexes of cold tolerance of evergreen broad-leaved plants. References
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［16］ MITTLER R. Oxidative stress,antioxidants and stress tolerance［J］.Trends in Plant Science, 2002, 7(9): 405-410. ［17］ VERBRUGGEN N. Osmoregulation of a pyroline-5-caboxylate reductase gene in Arabidopsis thaliana［J］. Plant Physiol, 1993(109): 771-781.
［18］ JIANG H, XU YC, LI YR, et al. The study on cold resistance of different Citrus medica strains［J］. Acta Horticulturae Sinica, 2012, 39(3): 525-532. (in Chinese)
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［Methods］Three species of evergreen broad-leaved woody plants, i.e., Ligustrum lucidum, Ilex cornuta and Eriobotrya japonica, were selected as the experimental materials. The morphological performances and the changes of the physiological indexes were observed and measured during the overwintering period in the open field in Beijing. The relationship between the indexes and the low temperature was also analyzed. The strength of cold tolerance of the three species was compared.
［Results］ The electrical conductivity, the contents of MDA and proline were negatively correlated with the corresponding low temperature. The contents of soluble sugar and soluble protein increased with the dropping temperature, but they had little response to the short-term temperature rise.
［Conclusions］Combined with morphological and physiological indexes, it was found that the changes of the contents of proline and soluble sugar among the physiological indexes were closely related to the cold tolerances of the three tree species of broad-leaved woody plants. The cold tolerance of I. cornuta was the strongest, E. japonica was the second, and that of L. lucidum was the worst.
Key words Evergreen broad-leaved woody plant; Cold tolerance; Overwintering
Received: January 12, 2021 Accepted: March 6, 2021
Supported by Key Project of Beijing Municipal Education Commission; Project of Construction of Advanced Horticulture Under Beijing Municipality (2020); National Natural Science Foundation of China (31201645; 31640070); Beijing Municipal Natural Science Foundation (3172006); Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT 20150503).
Sifan LI (1997-), female, P. R. China, Master, devoted to research about cultivation physiology of ornamental plants.
*Corresponding author. E-mail: shenman@vip.sina.com.
Beijing is located in the subhumid area of the warm temperate zone, and has a typical continental monsoon climate. Its natural zonal plant communities are mixed coniferous and deciduous broad-leaved mixed forests. The woody plants in such natural communities consist of a large number of deciduous trees and shrubs and a small number of evergreen coniferous trees. The composition of such natural communities determines that conifers and deciduous broad-leaved trees are the main landscaping tree species in Beijing, and evergreen broad-leaved trees are rarely used. Therefore, compared with the three seasons of spring, summer and autumn, Beijings winter greening landscape is monotonous and lacks vitality［1］. If some southern evergreen broad-leaved trees with high ornamental value and able to withstand the low temperature in winter in the north can be introduced to Beijing, they will not only enrich the tree species diversity in the urban garden landscape, but also optimize the landscape community structure of the garden plants in the northern region and improve the ecological environment of the northern region. The urban landscaping department has been promoting the northward migration of evergreen broad-leaved plants. However, based on the current introduction, the types of evergreen broad-leaved tree species in Beijing are still relatively few and it is difficult to promote new varieties. The main reason is the geographical environment, especially the low temperature in winter, which severely restricts the normal growth of evergreen broad-leaved trees in northern regions［2］.
The climate of Beijing is characterized by dry and cold winter with low rainfall, low humidity, large temperature difference between day and night and strong wind, and dry and windy spring with a temperature rising rapidly. This condition will cause repeated freezing and thawing of plant leaves and thus damage. Many evergreen tree species that grow at latitudes higher than Beijing and lower temperatures than Beijing are difficult to be cultivated in Beijing［3-5］. In the work of introducing evergreen broad-leaved plants, it was found that due to the large annual changes in natural climate conditions, short-term introduction success is not enough to support the introduction of tree species to adapt to local natural conditions, and the inadaptability of some tree species to low temperature cannot be observed in a short period of time. Therefore, it is very necessary to observe the evergreen broad-leaved plants introduced in northern regions for a long term and study their low temperature tolerances.
Based on the above research background, in this study, three common evergreen broad-leaved trees in East China were introduced into Beijing to observe the differences in plant leaf morphology during the natural overwintering process, analyze the changes in physiological indexes related to cold tolerance and their correlation with natural cooling, and compare the strength of cold tolerance of the three tree species, so as to provide experimental reference basis for establishing evaluation indexes of cold tolerance of evergreen broad-leaved plants and selecting cold-tolerant evergreen broad-leaved tree species suitable for planting in Beijing area.
Materials and Methods
Experimental materials
The plant materials are Ligustrum compactum, Ilex cornuta, and Eriobotrya japonica seedlings with a larger soil balls introduced from Yanling County, Xuchang City, Henan Province (belonging to the northern subtropical evergreen broad-leaved plant distribution area) in April 2002. They were planted in the office area and family residential area of the Fangshan Highway Branch in Liangxiang West Road of Fangshan District, Beijing. The plants were located in the planting area with good microclimate conditions (leeward and sunny, 2 to 4 m away from the buildings on the south and west sides of the buildings). At the early stage of introduction (2002-2006), winter was supplemented with cold-proof measures, that is, blue plastic film cold-proof sheds were used, grass ropes were wrapped around the trunk and main branches of the plants, and the base of the trunk was cultivated. Conventional water and fertilizer cultivation management was adopted. Experimental methods
Leaf shape observation
From November 2015 to April 2016, L. compactum, I. cornuta, and E. japonica at the Fangshan test site were observed and photographed every 2 weeks, and the morphological indexes of plant leaves were recorded. The daily weather conditions were recorded according to the weather forecast. The daily average temperature and daily minimum temperature in a small environment were measured using a GGL-20 temperature and humidity automatic recorder, and the monthly average maximum temperature and monthly average minimum temperature were calculated.
Determination of physiological indexes
Mature leaf samples were collected at 10:00 am throughout the winter (November 2015 to March 2016) for the determination of physiological and biochemical indexes. L. compactum and E. japonica leaves could not be sampled from February to March 2016 due to browning and withering. The specific sampling time were as follows: ① early winter, the stable temperature period: November 19, 2015, ② the sudden temperature drop period: November 28, 2015 (on November 22, there was a heavy snowfall in Beijing area, and the temperature dropped sharply, so the sampling was increased once in November, and noted in the figure as "11t"), ③ the period of gradual decrease in temperature: from mid-December 2015 to mid-January 2016, and ④ the period of warming in early spring: mid-February to mid-March 2016.
The various physiological indexes were carried out according to the method of Li［6］. The relative conductivity was determined by the conductivity method; the malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) method; the proline content was determined by acid ninhydrin method; the anthrone colorimetric method was used for the determination of soluble sugar content; and the Coomassie brilliant blue method was used for the determination of soluble protein content.
Statistics and analysis
The experimental data were analyzed by variance analysis and graphed using Microsoft Excel, GraphPad Prism 7.0 and SPSS 17.0 software.
Results and Analysis
Changes in leaf morphological indexes of three evergreen broad-leaved trees during overwintering
Plant leaves showed the most obvious changes during overwintering. From October 2015 to April 2016, the changes in leaf color and leaf shape of these three evergreen broad-leaved plants were observed and recorded (Fig. 1). As the winter temperature dropped, the leaves of the three tree species were brownish and slightly curled and turned. At the extreme low temperature on January 3, 2016, the leaves of the three tree species deeply browned, suffered from more serious water loss and even fell off. In the deep winter of February, the leaves of L. compactum were all browned, dried, and easy to fall off. By March, the leaves on the annual branches fell off and the branches became bald. From December, part of the leaves of I. cornuta were browned but the plants were normal as a whole. By March, the leaves were partially curled and dried, and the fruit was all browned, and more likely to drop. For E. japonica, only a small amount of leaves changed their color in January, the plants were normal as a whole, and the leaf color still changed less until March. As the temperature rose in early spring, the branches of L. compactum, I. cornuta and E. japonica grew out new leaves one after another in April.
Combined with the observation results of leaf texture, it was found that the leaves of L. compactum were thin, the leaves of I. cornuta were hard and leathery, and the leaves of E. japonica were covered with hairs. We believes that during the overwintering period in the open field, as the temperature drops, the leaves of L. compactum suffer from frost damage because of their thin texture, while the leaves of E. japonica are protected by hairs, can relatively reduce water loss and suffer less frost damage, and because of the wax on the leaf surface, I. cornuta is least affected by low temperature. From the perspective of leaf morphology, the order of cold tolerance of the three evergreen broad-leaved tree species was: I. cornuta>E. japonica>L. compactum.
Changes in physiological indexes of three evergreen broad-leaved plants during overwintering
Changes in relative conductivity and malondialdehyde content of leaves during overwintering
The different letters mean significant differences (P<0.05). The same below.
Sifan LI et al. The Comparison of Cold tolerance of Three Species of Evergreen Broad-leaved Woody Plants
Cell membrane is the primary part of plants that is damaged by low temperature［7］. The conductivity of the leaves can reflect the degree of chilling damage to the leaves of plants. The relative conductivity of plants is usually determined to reflect the degree of freezing damage to plants. The principle is that when plants are severely frost-damaged, a series of dynamic changes will occur in the cell membrane, leading to the exosmosis of electrolytes in plant cells［8］. It can be seen from Fig. 2 that as the temperature decreased, the conductivity of the leaves of the three evergreen tree species increased, and when the temperature rose, the electrical conductivity decreased again, which was consistent with the law of temperature changes. Specifically, at the sudden temperature drop on November 28, 2015, the conductivity values of the leaves of the three tree species all rose rapidly, and were significantly higher than those in other time periods. By December, the conductivity of the three tree species adapted to the continuous low temperature decreased again to the original level. During the whole overwintering process, the conductivity of I. cornuta and L. compactum changed significantly between different months, indicating that the conductivity changes were closely related to low temperature, while the value of E. japonica did not change significantly.
MDA content is the product of membrane lipid peroxidation, and the change of its content reflects the degree of damage to plants, and is one of the important signs of plant membrane damage by adversity［9］. Therefore, determining the MDA content is an effective method to determine the cold resistance of plants.
Fig. 2 shows that the MDA contents of the three broad-leaved plants all increased with the decrease of temperature, and all reached their maximums in December 2015. After a sharp drop in temperature on November 22, the MDA content of I. cornuta rose significantly, reached the highest value in December, and then slowly decreased, indicating I. cornutas response to the sudden drop in temperature. The change trends of MDA content in L. compactum and E. japonica were similar to that of I. cornuta. They reached the highest value in December and then decreased, but the increase rates of the two were higher than that of I. cornuta. The decreases of the MDA contents in the leaves of L. compactum and E. japonica in January might be due to the browning and drying of the leaves. In November, the MDA content of I. cornuta was significantly lower than those of L. compactum and E. japonica, indicating that I. cornuta had better cold tolerance than the former two.
Changes in proline and malondialdehyde contents of leaves during overwintering
The decrease in water content of plant tissues after being subjected to low temperature stress is conducive to the accumulation of dry matter in the body, which leads to the increase of cold resistance of plants［10］. Fig. 3 shows that during the natural temperature declining process, the leaf water contents of the three broad-leaved plants showed an overall downward trend. Among them, the water content of E. japonica dropped rapidly when the temperature suddenly dropped on November 22, and then showed an upward trend in December. I. cornuta and L. compactum showed a continuous downward trend during the entire temperature declining process, and the water content of I. cornuta rose when the temperature rose in March. The leaf water contents of the three tree species were negatively correlated with temperature changes. Proline is an important protective substance for cold resistance. Because of its strong hydrophilicity, it can stabilize the metabolic processes in protoplast colloids and tissues, thereby reducing the freezing point of cells, so its content is related to the cold resistance of plants［11］. Fig. 3 shows that the proline contents of the three evergreen broad-leaved tree species all increased with the decrease of temperature, and when the temperature rose, the proline content of I. cornuta decreased again. Except for the highest proline content of E. japonica, which appeared in December, the highest proline contents of L. compactum and I. cornuta appeared in January 2016. During the sudden temperature drop in November 2015, the proline contents of E. japonica and L. compactum showed no significant changes.
From the perspective of the overall change trend, the change values of proline content were arranged from large to small as: I. cornuta﹥E. japonica﹥L. compactum, and the differences were significant.
Changes in soluble sugar and soluble protein contents of leaves during overwintering
Soluble sugars are important metabolites in plants. After being stressed, plants will actively accumulate some soluble sugars to reduce osmotic potential and freezing point, so as to adapt to changes in external environmental conditions［12］.
Fig. 4 shows that the soluble sugar contents of these three evergreen broad-leaved plants generally increased gradually as the temperature decreased. Due to the sudden temperature drop on November 22, the soluble sugar contents of the leaves of the three broad-leaved tree species increased sharply between 11t and December. L. compactum reached its maximum value in December, and afterwards, a large number of leaves were browned due to severe freezing damage, so the soluble sugar content of leaves decreased in January. The soluble sugar content of E. japonica leaves reached a maximum in January 2016. The soluble sugar content of I. cornuta showed an overall upward trend, and reached its maximum in February. And compared with other two tree species, the soluble sugar content of I. cornuta was relatively high, so it was inferred that I. cornuta had stronger cold tolerance.
Regarding the relationship between soluble protein and plant cold resistance, some researchers believe that soluble protein is highly hydrophilic, and can increase the water holding capacity of cells and reduce damage caused by icing, thereby improving the cold resistance of plants［13］. It was found from this study that the soluble protein contents of the three evergreen broad-leaved plants showed an upward trend as the temperature dropped. The soluble protein contents of L. compactum and E. japonica reached their maximums in December during the low temperature period from the temperature decrease in November 22 to December (Fig. 4). From the overall change trend, the maximum soluble protein contents of I. cornuta, E. japonica and L. compactum all appeared in January 2016, and they were negatively correlated with sub-zero low temperature in the deep winter (from later November to January). During the whole winter temperature declining process, the differences between the various tree species changed significantly, and the soluble protein content changes ranked as L. compactum﹥I. cornuta﹥E. japonica. Conclusions and Discussion
During the overwintering period of plants in the open field, in addition to measuring the relevant physiological indexes of plants to judge their cold tolerance (resistance), it is also necessary to observe the growth status and morphological performance of the plants during the overwintering process. Wan et al.［5］ observed the overwintering performance of evergreen broad-leaved plants such as Buxus megistophyll, B. sinica, Ligustrum lucidum, Pyracantha fortuneana, Yucca gloriosa, Photinia serratifolia. and Hedera nepalensis var. sinensis by the above method and identified the cold resistance of these plants. In this study, we also observed the overwintering performance of 3 evergreen tree species introduced in Beijing and cultivated in Fangshan area. According to the morphological changes of each tree species, it was found that the different texture and structure of the leaves would affect their cold resistances, and during the winter, L. compactum turned from evergreen to semi-deciduous through leaves to adapt to the low temperature and drought in winter in Beijing.
During the overwintering period, the changes in the external morphology of plants can only show the response of plants to low temperature to a certain extent, and cannot quantify the cold tolerances of plants. Therefore, it must be combined with the measurement of physiological indexes to make a comprehensive judgment. Plant cells communicate with the natural environment through their cell membranes, and the impact of low temperature stress on plants first acts on the plasma membrane. When plants are injured by low temperature, the unsaturated fatty acids in the membrane will undergo membrane lipid peroxidation, and the final product MDA accumulated will damage the cell membrane and cause electrolyte leakage. Therefore, measuring the conductivity value and MDA content of plants can reflect the degree of damage to the cell membrane and the degree of cold resistance of plants［14-15］. After plants are injured by low temperature, the lower the relative conductivity and MDA content, the stronger the cold tolerance of plants; otherwise, the weaker the cold tolerance［16］. In this study, it was found that the conductivity of L. compactum during the overwintering period was the highest, and its cold resistance was the worst; and the MDA contents of L. compactum and E. japonica leaves were higher than that of I. cornuta, indicating that the cold resistance of I. cornuta was stronger than that of L. compactum and E. japonica. Proline is an important cold-resistant protective substance in plants. When the temperature decreases, its content increases rapidly, indicating that plants cold resistance is enhanced. When the adversity is relieved, the content of proline decreases rapidly［17］. Jiang et al.［18］ compared the cold resistances of different strains of Citrus medica and found that the proline contents of the strains with strong cold resistance maintained a higher level. Therefore, the strength of cold resistance of plants can be judged by measuring the content of proline. In this study, the proline content of the leaves of the three tree species all increased with the decrease of temperature. The changes of the proline content ranked as I. cornuta﹥E. japonica﹥L. compactum, indicating I. cornuta had the strongest cold resistance, followed by E. japonica and L. compactum was the weakest, which was consistent with the results obtained by morphological observation.
As cell contents, soluble sugar and soluble protein are also important protective substances for cold resistance of plants, which can maintain the structural stability of plant cells, regulate osmotic pressure, and play a positive role in resisting cold damage to plants［19］. Under low temperature stress, plants have been proved by a large number of experiments by increasing the content of soluble sugar in the body to improve cold resistance. Therefore, the contents of soluble sugar and soluble protein can be used to identify the cold resistance of plants［20-23］. It is generally believed that the content of soluble protein increases with the increase in cold resistance under low temperature stress, but there are also reports pointing that the increase in the content of soluble protein in some plants has no correlation with their cold resistance［24］. In this study, the soluble sugar content of I. cornuta was higher than that of E. japonica and L. compactum, indicating that the cold tolerance of I. cornuta was higher than that of E. japonica and L. compactum. From the perspective of changes of soluble protein contents in the leaves of the three tree species, L. compactum had the largest change, followed by I. cornuta, and E. japonica had the smallest change.
To sum up, during the whole wintering period of the three evergreen broad-leaved trees, there was certain correlation between the measurement results of various physiological indexes and the natural low temperature and the cold tolerances of the tree species. However, the cold tolerance of evergreen broad-leaved plants cannot be measured by a single physiological index, but instead, a comprehensive analysis should be carried out among various physiological indexes, combined with plant morphological indexes, tree species characteristics and complex environmental factors. In the analysis of various indexes in this study, it was found that the proline content and soluble sugar content were closely related to the cold tolerances of the three evergreen broad-leaved trees and also consistent with the observation results of the morphological changes of the plants during overwintering, and thus can be screened as indexes of cold tolerance of evergreen broad-leaved plants. References
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