Effects of Low Temperature on Physiological Responses of Two White Clover Cultivars

Li Xun, Wang Peng, Xie Fu-chun, Sun Hua-shan, Liu Ke-meng, Guan Tong, Fu Zhen-xuan, and Chen Ya-jun

College of Horticulture, Northeast Agricultural University, Harbin 150030, China

Introduction

Temperature directly affects plant transpiration,metabolism, growth and development and almost all the enzymatic reactions. Low temperature can inhibit the normal growth of plants, affect their production performance and economic traits, and even cause plant death (Penget al., 2007). When plants are exposed to low temperature stress, the plant cells usually regulate the related metabolic pathways through their own defense network, generating osmotic adjustment substances to reduce or eliminate the damage caused by low temperature stress. Soluble sugar, free proline,malondialdehyde (MDA), electrical conductivity and so on are closely related to low temperature stress injury, usually as indicators of plant stress resistance,whose contents and varied trends to a certain extent can explain the degree of plant damage and cold resistance (Sun and Zhang, 1990). For example, the higher the content of soluble sugar and free proline accumulate in cells, the stronger the cold resistance of the plants is (Yanget al., 2015).

Vegetative ground cover plants are important parts of human living environment components which can purify the air, prevent soil erosion, reduce noise and adjust the atmospheric temperature and humidity.White clover is a perennial cold season ground cover belonging to the family of Leguminosae. It is worldwide distribution and possesses strong ability to adapt to the cold environment. Even under the condition of -15℃, white clover can still safely pass the winter. In the last decades, it has been used not only as an excellent legume forage, but also as an important high-quality urban greening grass. However,some white clover cultivars introduced from other regions of the world are not adapted to the domestic low temperature environment, such as in Heilongjiang Province in China, resulting in weak growth, poor wintering rate, thus reducing their ornamental values,which is also a major limiting factor for their extensive applications in China. In order to solve this problem,the important measurement is to improve white clover cold resistance and increase its adaptation ability to the ecological environment in lower temperature areas.Therefore, the objectives of this study were to provide basic data for the evaluation of cold resistance with different genotype white clovers from different regions of the world, and to reveal the physiological response mechanisms to lower temperature within cultivars.

Materials and Methods

Plant materials

Two kinds of genotype cultivars of white clover(Trifolium repens L.) called Koala and Longping No.1 were used in this study. Koala (represented by K) was a native white clover in Australia from south hemisphere and was provided by the Ministry of Agriculture Grass Industry Company, Beijing, China,while Longping No.1 (represented by L) was a native landrace in the northeast of China, Heilongjiang Province, and was provided by Northeast Agricultural University. Koala and Longping No.1 possessed different phenotypes according to their morphological characters. The height of Koala was about 13.0-15.0 cm, and the average leaflet area was about 296±8 mm2,while the height of Longping No.1 was about 8.0-10.0 cm, the average leaflet area was 112±5 mm2, which were significant lower than those of Koala.

Methods

The experiment was conducted in the greenhouse of Horticultural Experimental Station in Northeast Agricultural University from August, 2017 to December,2017. Seeds were sown in seedling boxes (PVC box,30×70×10 cm) with sowing rate of 10 g · m-2. One month later, the seedlings were transplanted into seedling pots (20 cm in diameter and 15 cm tall), and with the soil 2.0 kg. The content of soil organic matter was 15.88 g · kg-1, and pH was 7.0. When the seedlings height reached to 5 cm, each pot was saved 10 plants.After two and a half months of normal management,the seedlings were pretreated in a 2℃ refrigerator for 24 h, and then subjected to a series of low temperature treatments for 1 h. The treatment temperatures were set to six gradients in turn at 25℃, 10℃, 5℃, 0℃,-5℃ and -10℃. Then the seedlings above ground were harvested for physiological indexes measurements.Free proline, soluble protein, soluble sugar (SS),malondialdehyde (MDA), starch (S), superoxide dismutase (SOD) activity and electrical conductivity(EC) of two kinds of white clovers were determined with the methods discribed by Haoet al(2004).

Results

Effects of low temperature on SOD, soluble protein and free proline content

The activity of SOD in the two cultivars of white clover increased along with the decrease of the treatment temperature, and the trends of SOD increased the fastest in the temperature ranged from 10℃ to 5℃. The difference in SOD activity of Longping No.1 was not significant, when the temperature was reduced from 25℃ to 10℃ (Fig. 1a).The change trend of soluble protein was consistent within the two cultivars under low temperature stress(Fig. 1b) with increases first and then decreases. When treated at 10℃, the content of soluble protein reached the highest amount for both cultivars. There was no significant difference in the content of soluble protein in Koala, when the temperature decreased from 5℃ to 0℃, but Longping No.1 just had significant difference from 0℃ to -5℃. The content of free proline in plants increased, during all the treatment periods. There was no significant difference in free proline contents of Koala, when the temperature was decreased from -5℃ to -10℃. During the whole process, the indexes of Koala were lower than those of Longping No.1. Except for SOD activity, two cultivars had no significant differences above 5℃. The contents of free proline and soluble protein in the two cultivars were the most significantly different at -10℃, and Longping No.1 was 42.6% and 40.3% higher than those in Koala.

Fig. 1 Effects of low temperature stress on SOD, soluble protein and free proline contents in the two white clover cultivars

Effects of low temperature on MDA content and electrical conductivity

As shown in Fig. 2, both electrical conductivity and MDA in the two white clover cultivars were increased,during the whole low temperature stress. The electrical conductivity in Koala increased rapidly after 0℃, and its electrical conductivity was significantly higher than that of Longping No.1, while the electrical conductivity increased slowly from 0℃ to 25℃. The electrical conductivity and MDA content of Koala were always higher than those of Longping No.1 under different low temperature treatments. MDA contents of the two white clover cultivars reached significant difference at 0℃, and the content of MDA in Koala was 58.6%higher than that in Longping No.1, the electrical conductivity of the two white clover cultivars had significant difference at -10℃, and the electrical conductivity in Koala was 48.6 % higher than that in Longping No.1.

Effects of low temperature on starch and soluble sugar contents

As shown in Fig. 3, under low temperature stress,the contents of soluble sugar in the two white clover cultivars increased with the decrease of treatment temperature, but starch in the two cultivars decreased.The contents of starch and soluble sugar in Koala were always lower than those in Longping No.1 under different low temperature treatments. When treated at-5℃, soluble sugar content of Koala increased slowly than that of Longping No.1. When treated at 5℃, the trend of starch content decreased slowly. The soluble sugar contents of the two white clover cultivars reached the most significant difference at 0℃, and the content of soluble sugar in Longping No.1 was 8 % higher than that in Koala.

Fig. 2 Effects of low temperature stress on MDA content and electrical conductivity in the two white clover cultivars

Fig. 3 Effects of low temperature stress on starch and soluble sugar contents in the two white clover cultivars

Discussion

Plants under low temperature stress, often through their own defense networks to regulate the metabolic pathway, produce osmotic conditioning substances to reduce or eliminate stress damage. The effects of low temperature stress on physiological responses of white clover seedlings were examined in this study.

Osmotic adjustment substances under low temperature

Usually under the environment of stress (drought,high temperature, low temperature, salt, etc.), the cells would take the initiative to form some osmotic adjustment substances, passively lose some water,so as to improve the solute concentration, reduce water potential, and ensure that the cells continued to absorb water from the outside to maintain their normal growth. This active adaptation of plants to stress was called osmotic adjustment. Soluble sugar accumulation at low temperature improved the osmotic potential, was conducive to cell osmotic adjustment and protected the structure of the membrane, therefore, soluble sugar played an important role in plant cold resistance (Wuet al., 2008). Soluble protein, soluble sugar and free proline were important osmotic adjustment substances in plant cells, and the soluble protein had strong hydrophilicity. The increase of the content of soluble protein led to the increase of bound water content in the cells and the improvement of the water retention capacity of the cells, meanwhile, the intracellular concentration was increased, and the possibility of ice formation was reduced. Soluble protein content was one of the important indexes in plant cold resistance.Most researchers believed that the increase in plant soluble protein content under low temperature stress could significantly enhance cell water holding capacity and reduce the damage of protoplasts due to freezing (Luet al., 2012; Chinnusamyet al., 2003).Low temperature stress could increase the content of soluble protein in root cells, effectively alleviate cell metabolism and resist low temperature (Gao, 2014).Free proline was a kind of amino acid with strong hydration ability, which was considered to be the product of plants under stress. The increase of free proline content was beneficial to cell water holding and the stability of biological macromolecules (Zhanget al., 2017). Researches showed that plants produced large amounts of organic small molecules, such as sorbitol, betaine, glycerol and proline, when they were exposed to low temperature stress to adapt to changes in osmotic pressure inside and outside cells(Samiset al., 2002). Under low temperature stress,the dynamic changes of starch content in plant tissues could reflect the trend of carbohydrate accumulation,and a large degree of starch content reduction would lead to the decrease of the total carbohydrate contents(Castonguayet al., 1995). In this study, the contents of free proline and soluble sugar in the two varieties were positively correlated with the intensity of low temperature stress. It was suggested that the low temperature induced the activity of hydrolases,accelerated the decomposition of starch, increased the contents of free proline and soluble sugar rapidly,increased the cell liquid concentration and enhanced the frost resistance, which indicated that the contents of free proline and soluble sugar were closely related to cold resistance. With the decrease of temperature,soluble protein content decreased, indicating that soluble protein content was not only affected by low temperature, but also might be restricted by other factors. The content of starch had a negative correlation with the intensity of low temperature stress, which indicated that the two white clover cultivars could adapt to low temperature stress actively to degrade starch content to increase resistance and maintain high osmotic potential in the cells. The results in the present study were consistent with previous reports. The content of each index of Koala was always lower than that of Longping No.1. The stress injuries of low temperature on Longping No.1 were also always lower than those of Koala. This phenomenon might due to these two cultivars were produced in different temperature regions, resulting in different cold resistances in response to physiological changes.

Plant cell physiological protection under low temperature

Cell membrane was the interface and barrier between living cells and environment. The influence of various adverse environment on plant often acted on cell membrane first, changed its permeability, and the size of cell membrane permeability could reflect the degree of damage of cytoplasmic membrane. An important aspect of characterizing the permeability of plant cell membrane during electrolyte leakage was that the increase or decrease of electrical conductivity directly reflected the membrane permeability, and then showed the damage degree of structure and function of cell membrane under low temperature stress. The greater the electrical conductivity was,the better the permeability of cell membrane had, the weaker the cold resistance of plants was (Guet al.,2014). SOD was one of the most important active oxygen scavenging enzymes in plant cells, membrane lipid peroxidation could damage the enzymatic and non-enzymatic defense system, if the intensity of stress had not yet exceeded its own adaptability and resistance, plant cells would actively activate the antioxidant system, accelerate the synthesis of antioxidant protective enzymes or improve the activity of these enzymes, and speed the scavenging free radicals and active oxygen, thus reduced membrane damage. If the stress intensity exceeded the plant body's ability to withstand, low temperature damage to the plant was more and more serious, the plants could not resist the damage by increasing the activity of SOD and other enzymes, and the activity of SOD would be reduced (Daiet al., 2007). Under low temperature stress, the increase of reactive oxygen species in plants resulted in the accumulation of MDA in membrane lipids. MDA could crosslink biological macromolecules, such as lipids, nucleic acids, proteins and sugars, which further damaged the structure and function of plasma membrane and increased the extravasation of protoplasts. The amount of MDA content could reflect the size of membrane lipid peroxides, that was, the extent of damage (Xuet al.,2015). In this study, under low temperature stress, the electrical conductivity and activity of SOD and MDA in the two cultivars increased slowly in the early stage,that was to say, at this temperature, the plants could still grow normally. When the temperature reduced to a certain temperature, the growth rate was rapidly decreased, which indicated that the damage of the membrane system was obviously increased, but it did not cause irreversible lethal damage under this temperature condition, the growth of plants had been more difficult, and could only barely sustain life activities.

Conclusions

To sum up, white clover developed a variety of physiological strategies to deal with low temperature stress, but the cold resistances were different between the two cultivars in this study. Comprehensive assessments on the physiological responses indicated that the cultivar Longping No.1 had strong ability of osmotic regulation and osmotic protection than Koala, and it maintained better membrane permeability, suggesting that Longping No.1 had strong cold resistance than Koala, which could be due to the two cultivars were produced in different temperature regions, resulting in different cold resistances in response to physiological changes. These results would provide helpful information for further improving white clover cold resistance in practical breeding program.

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