Effect of Chilling Stress on Synthesis of Antioxidant Enzymes, Osmotic Adjustment Substances and Membrane Lipid Peroxide Levels in Two White Clover Cultivars

Li Xu, Liu Hao-yue, Wang Li-na, Zhao Yi-hang, Hu Yao, Zhang Xiao-meng, Meng ling-dong, and Yin Xiu-jie

College of Animal Sciences and Technology, Northeast Agricultural University, Harbin 150030, China

Abstract: Although white clover has been widely studied and applied, less is known about its physiological responses to chilling stress in northern China.The effects of acclimation time of chilling stress in the two white clover cultivars, Rivendel and Haifa, after exposure to 4℃ for 0, 4, 8, 12, 24 and 48 h were investigated.Seven physiological indexes were measured in shoots of the two cultivars.It was found that Rivendel had a strong ability to maintain the stability of cells, cope with oxidative damage and accumulate osmotic adjustment substances compared with Haifa.In addition, a comprehensive evaluation on the chilling tolerance of the two cultivars based on subordinate function value analysis showed a stronger chilling tolerance of Rivendel than Haifa.

Key words: chilling stress, physiological index, white clover

Introduction

White clover (Trifolium repensL.), a perennial herb, has a long cultivation history and is currently a sought-after herbage in livestock.White clover has a wide distribution range, strong adaptability to a low temperature environment and high application value (Qu and Wang, 2020).It is used as a forage worldwide (Lianget al., 2020), soil nutrition improvement features for greening and is planted in forest orchards (Wanget al., 2017).White clover is an important economic plant in Europe, South America and New Zealand.It was introduced to China in the 1920s.White clover has also been widely planted and used in many areas (Yanget al., 2016).However, chilling stress is the key factor limiting the introduction of white clover.

Low temperature stress within 0-10°C can cause chilling injury in cold resistance research of south China rubber (Cui, 1999).It is associated with damaging the plant membranous structure and biological enzyme activity, which can directly affect the metabolism, growth and enzymatic reactions of plants.For example, when the biofilm of cells is damaged, the cells begin to dehydrate, the permeability of the plasma membrane is increased, sugar and protein are transformed and hydrolyzed, the metabolism of reactive oxygen is maladjusted, the physiological dynamic balance is broken and metabolic activities cannot be conducted in an orderly way.Furthermore, it can also affect the production performance and economic characteristics of plants and even cause plant death (Maet al., 2020).Membrane stability is correlated with plant chilling tolerance (Valizadehet al., 2018).Free proline, soluble sugar and soluble protein are often used as chilling tolerance indexes, because they are effective in preventing death from chilling in the cells of plants (Zhang, 2019).Accumulation degree of malondialdehyde (MDA), an end product of membrane lipid peroxidation, is considered to be a primary standard to detect the degrees of membrane lipid oxidation under chilling stress (Liet al., 2020).Previous researches have shown that antioxidant enzymes play a vital role in improving chilling tolerance.Superoxide dismutase (SOD), peroxidase (POD) and oxygen anion (O2-) play a significant role in chilling tolerance (Zhouet al., 2019).Low temperature can affect the period of seedling establishment of white clover, which can influence its development and utilization (Renet al., 2018).White clover planted in the north of China has faced many problems, such as a low wintering rate, susceptibility to low temperature and normal growth only in the first year of planting, which greatly limits the introduction of white clover to northern China (Qu and Wang, 2020).Some kinds of white clover cultivars cannot be introduced to a low temperature environment, because low temperature can result in weak growth and a poor wintering rate, which can reduce the use of white clover (Liet al., 2018).

To introduce suitable cultivars to China's northern areas, this study determined the effects of different durations of chilling stress on the physiology of the two white clover cultivars, Rivendel and Haifa.This research would provide a primary basis for breeding new tolerant white clover cultivars to chilling stress.

Materials and Methods

Plant materials

Rivendel and Haifa, two widely planted white clover cultivars, were collected from the seed bank of the Institute of Prataculture Research of Chinese Academy of Agricultural Sciences.This study was conducted in the greenhouse of Northeast Agricultural University.Seeds of the two white clover cultivars with full grains and uniform size were selected for seedling cultivation.These seeds were washed in distilled water, surface-sterilized in a 6% NaClO solution for 15 min and rinsed three times in sterile distilled water.Then, they were sown into trays with holes containing the soil.The soil organic matter content was 15.88 g • kg-1, and pH was 7.0.Five seeds were sown into each hole on 12 December, 2017.A piece of sponge was placed in the bottom of the hole tray to prevent grit from leaching out.These trays were placed in a controlled environment with temperature of 25℃ and a relative humidity of 65%.The photosynthetically active density was 400 μmol•m-2•s-1, with 12 h light and 12 h dark.The seedlings were watered every two days and irrigated two times per week with Hoagland nutrient solution.

Experimental treatments and measurements

After 30 days of growth, seedlings that were used for the experiments were removed from the culture greenhouse, rinsed with distilled water and then rinsed three times with deionized water, wrapped with plastic film and placed in separate sealed plastic bags for the chilling treatments.All the experimental materials were placed into the refrigerator, which caused the internal temperature of the sample to drop from 25℃to 4℃.The average degrading cooling rate was approximately 2.5℃ • h-1.The chilling treatments were initiated, when the temperature reached 4℃.The processing time gradients were 4, 8, 12, 24 and 48 h at 4℃.For the control, plants were kept at 25℃ in the incubator.The seedlings were harvested at each time point, and then the seedling shoots were separated by scissors and collected for physiological index measurements.These materials were immediately frozen in liquid nitrogen and stored at -80℃ for further physiological measurements, including O2-, MDA, SOD, POD, free proline, soluble sugar and soluble protein according to Haoet al(2004).

Data analysis

In this experiment, seven physiological indexes were measured at different treatment times, and a comprehensive evaluation of the chilling tolerance of these two cultivars was performed based on subordinate function value analysis.The calculation equation of the membership function value [X(u1),X(u2)] was analyzed as the followings (Cui, 2006):

Xwas the measurement value of a certain index of a certain cultivar;Xmaxwas the maximum value of this index; Similarly,Xminwas the minimum value.If the measured indexes were positively correlated to plant chilling tolerance, formula (1) was used to calculate the subordinate function value, while formula (2) was used for the negative correlation.The specific subordinate values of each cultivar's indexes were added, and the averages were computed and compared.The higher the average value, the stronger was the plant's tolerance to chilling stress.

Excel 2010 was used for data collation and chart making.IBM SPSS Statistics 19.0 was used for statistical evaluation.All the data were subjected to ANOVA, and if a significant (p<0.05)Fratio occurred for different treatment effects, an LSD was calculated.

Results

Differential O2- responses of two cultivars to chilling stress

O2-was one of the intermediate products of membrane lipid peroxidation, and the degree of membrane lipid peroxidation reflected the damage of the cell membrane.As shown in Fig.1, for shoots of the two white clover cultivars, O2-content increased with treatment time, and O2-content in Haifa maintained a high level compared with that in Rivendel, during the whole treatment process (p<0.05).Furthermore, the increase in O2-content in Haifa (22.48 nmol • min-1FW) was higher than that in Rivendel (18.41 nmol • min-1FW) after 48 h chilling stress.

Fig.1 Effects of chilling treatment on O2- content in shoots of Rivendel and Haifa

Differential MDA responses of two cultivars to chilling stress

MDA, one of the final products of membrane lipid peroxidation, could reflect damage to the cell membrane.As shown in Fig.2, under the control conditions, MDA content in Rivendel was significantly lower than that in Haifa (p<0.05).MDA content increased with treatment time, and in both cultivars, MDA content increased rapidly after 8 h (p<0.05).After 48 h of treatment, MDA content in Rivendel and Haifa increased by 11.22 and 11.59 μmol•g-1FW, respectively.In brief, MDA content in Rivendel increased at a slower rate than that in Haifa.

Fig.2 Effects of chilling treatment on MDA content in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Differential SOD responses of two cultivars to chilling stress

SOD was the first line of defense of plant antioxidant systems, and its level could reflect the ability to remove reactive oxygen species (ROS).As shown in Fig.3, the change trend of SOD activity was consistent in the shoots of these two white clover cultivars under the treatments and the activity increased first and then decreased.Interestingly, SOD activity peaked after 8 h of treatment in Rivendel (763.19 U • g-1FW) and after 12 h in Haifa (591.51 U • g-1FW).It could be concluded that the peak in the change trend in Rivendel (8 h) was earlier than that in Haifa (12 h).

Fig.3 Effects of chilling treatment on SOD activity in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Differential POD responses of two cultivars to chilling stress

POD was an important enzyme in membrane protection systems and could be an indicator of the ability to remove ROS.As shown in Fig.4, the activity of POD in the shoots of Haifa and Rivendel was observed to increase within the initial stage of treatment, but this tendency did not continue throughout all the processing.Within the initial 12 h, POD activity in Rivendel and Haifa increased by 81.17% and 64.11%, respectively.After 12 h, POD activity in Rivendel and Haifa decreased by 28.08% and 31.13%, respectively.It could be concluded that POD activity increased more rapidly and decreased at a slower rate in Rivendel than that in Haifa.

Fig.4 Effects of chilling treatment on POD activity in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Differential free proline responses of two cultivars to chilling stress

Free proline, an important substance accumulated in response to osmotic adjustment in plants, could endow plants with the ability to overcome adversity.As shown in Fig.5, free proline content in shoots of Rivendel first increased and then decreased and finally increased, showing an N-type trend.Free proline content increased rapidly up to 389.72 μmol • g-1FW at 8 h and peaked (450.02 μmol • g-1FW) at 48 h.However, the trend of free proline content in Haifa was different from that in Rivendel.Free proline content in Haifa peaked (267.68 μmol • g-1FW) at 12 h and then decreased.It could be concluded that the peak occurred earlier in Rivendel than that in Haifa.

Fig.5 Effects of chilling treatment on free proline content in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Differential soluble sugar responses of two cultivars to chilling stress

Soluble sugar had several beneficial effects in protecting plants against physiological stress, and its content in cells could reflect adaption to adverse stress.As shown in Fig.6, for shoots, soluble sugar content in Rivendel first increased to the highest (13.99 mmol • L-1• g-1FW) at 8 h and then decreased to 10.71 mmol • L-1• g-1FW at 24 h and finally increased to 12.44 mmol • L-1• g-1FW at 48 h.Soluble sugar content in Haifa first increased to 9.70 mmol • L-1• g-1FW at 8 h, then decreased to 8.33 mmol • L-1• g-1FW at 12 h, and finally increased to the highest 11.12 mmol • L-1• g-1FW at 48 h.This result showed that soluble sugar content in Rivendel peaked faster than that in Haifa.

Fig.6 Effects of chilling treatment on soluble sugar content in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Differential soluble protein responses of two cultivars to chilling stress

Soluble protein could protect plants from damage caused by chilling stress, and its content in cells reflected plant chilling tolerance.As shown in Fig.7, during the whole process, soluble protein content in shoots of these two white clovers first increased and then decreased and finally increased at the end of the process.The change trends of soluble protein showed an N-type shape.However, the peak in soluble protein content occurred at different time points in these two cultivars.Soluble protein content in Rivendel peaked at 48 h (3.15 mmol • L-1• g-1FW), while that in Haifa peaked at 12 h (2.86 mmol • L-1• g-1FW), but was still lower than that in Rivendel.

Fig.7 Effects of chilling treatment on soluble protein content in shoots of Rivendel and HaifaDifferent lowercase letters indicate significant differences (p<0.05) in the same cultivars between treatments; different capital letters indicate significant differences (p<0.05) in different cultivars in the same treatment.

Subordinate function analysis of two cultivars to chilling stress

According to Table 1, the data were arrayed in ascending order: Rivendel shoots (0.57)>Haifa shoots (0.52).It could be concluded that the cold tolerance of Rivendel was stronger than that of Haifa under chilling stress.

Table 1 Subordinate function values of shoots of Rivendel and Haifa during chilling process

Discussion

Membrane lipid peroxide under chilling stress

The cell membrane (also known as plasma membrane), a thin semipermeable membrane, surrounded the cytoplasm of a cell.The function of the cell membrane was to protect the cell by keeping harmful substances out and allowing certain substances into the cell (Heidarvand and Amiri, 2010).It could also act as a base of attachment for the cell wall.Thus, the cell membrane could also help the cell maintain its shape (Sunet al., 2020).The complete structure of the cell membrane was very important for cell growth.Chilling injury damaged the membrane structure of the cell, and it affected membrane permeability and the lipid composition of the plasma membrane (Alba-Jiménezet al., 2018).The content of O2-and MDA could reflect the degree of membrane lipid peroxidation (Zhouet al., 2019).

MDA, one of the final products of membrane lipid peroxidation, was toxic to the plasma membrane.MDA content could be considered the main factor of membrane damage (Nejadsadeghiet al., 2015).MDA could damage the cell membrane by increasing permeability, and a large amount of intracellular inorganic ions (K+) would undergo external flow, which caused the intracellular ion balance to be disrupted and then induced cellular metabolic disorder and finally the growth and development of plants were both affected (Chen, 1991).MDA not only reflected membrane damage, but also had a feedback effect (Chi and Liu, 2019).Thus, a change in MDA content was an indicator of the degree of damage (Zhanget al., 2020).Hence, MDA content in this experiment was measured to assess the degrees of chilling stress.In the present study, after 48 h of chilling treatment, the increase in MDA content was higher in Haifa than that in Rivendel.In other words, Haifa accumulated MDA faster than Rivendel under chilling stress.This finding indicated that Rivendel had a stronger chilling tolerance capability than Haifa.Cold-resistant cultivars could quickly accumulate MDA in many organisms, such as camellia (Zhouet al., 2019), wheat (Nejadsadeghiet al., 2015) and sweet cherry (Wuet al., 2019).

O2-was the intermediate product of membrane lipid peroxidation.Adversity could cause the accumulation of O2-, which could cause membrane damage (Zhouet al., 2019).The accumulation of O2-could reflect the cold tolerance of plants to a certain extent, and it was initially considered as an indicator of cold tolerance test (Xuet al., 2017).In the present study, chilling stress increased O2-content.This change tend was also confirmed by the observed increase in MDA content in shoots of Rivendel and Haifa under the same treatment.Liang (2016) found that the rate of free radical production in the leaves of Bermuda grass seedlings andZoysia japonicaseedlings increased significantly at low temperatures.Liuet al.(2016) found that O2-production rate of the leaves of cucumber seedlings increased under low temperature.In this study, O2-content was lower in Rivendel than that in Haifa, which indicated a lower chilling damage of Rivendel than Haifa.

Antioxidant enzymes under chilling stress

Oxidative stress was one of the major damages caused by low temperature stress, and the adaptability of plants to low temperature was closely related to their antioxidant ability.Oxidative stress was caused by reactive oxygen species (ROS), which interacted with many cellular components, causing oxidation reactions and major damage to plants (Sarvajeet and Narendra, 2010).Antioxidant enzymes could eliminate ROS in plants and maintain the balance of ROS and protective enzymes.Once the balance breaks, the accumulation of free radicals, membrane lipid peroxidation, and damage to cell structure and function would occur.Finally, the plant would be damaged or even die (Xuet al., 2017).SOD and POD were the main antioxidant enzymes in plant cells.

SOD was the first line of defense of plant antioxidant systems and the key rate-limiting enzyme, which could remove ROS from cells when plants were exposed to cold environments (Zhouet al., 2019).The main function of SOD was to clear O2-through an oxidation-reduction reaction (2O2-+2H+→H2O2+O2) (Fan, 2009).H2O2, the most stable form of ROS, was always taken as an indicator of oxidative stress; it could rapidly cross the cell membrane and influence the level of lipid peroxidation (Sarvajeet and Narendra, 2010).In the present study, SOD activity first increased and then decreased during chilling stress.The increase in SOD activity in the early stage of cold stress might be an adaptive response when plants were exposed to cold conditions, which could be beneficial for alleviating the oxidative stress caused by chilling stress.SOD activity in the shoots of Rivendel and Haifa decreased after 8 and 12 h, respectively, because chilling stress could cause membrane lipid peroxidation, which could damage antioxidant enzymes system.Furthermore, the peak SOD activity in the shoots of Rivendel appeared earlier, supporting its relatively fast adaptability to chilling stress.Zhouet al.(2019) also found that SOD activity in wild bluegrass root first increased and then decreased under overwintering period.Quet al.(2010) found that the peak SOD activity in cold resistant cultivars appeared early.

POD was an important enzyme in membrane protection systems and played an important role in scavenging free radicals and peroxidation products (Liet al., 2020).Under adverse conditions, superoxide anion radicals were produced in plant cells.SOD could scavenge superoxide anion radicals.SOD reacted with the superoxide anion to form H2O2and oxygen.POD could remove H2O2from cells (Zhanget al., 2019).When plants suffered from stress, POD could act synergistically with SOD to remove excess ROS, which could protect plants from damage.In this study, the increase in POD activity occurred more quickly in Rivendel than Haifa, and the rate of decrease in POD activity in Rivendel dropped slower than that in Haifa.It was suggested that Rivendel and Haifa were less and more sensitive to chilling temperature, respectively.Maet al.(2018) and Zhanget al.(2020) found that in cold resistance research of walnut and honeysuckle, a high level of POD activity was beneficial for plant tolerance to cold stress.

Substances for osmotic adjustment under chilling stress

Under adverse conditions, plants could accumulate many substances for osmotic adjustment, which could endow a cultivar with osmotic adjustment capabilities.Under stress conditions, cells could actively accumulate solute, which could lead to decreases in cell osmotic potential (Linet al., 2018).Free proline, soluble sugar and soluble protein were several important osmotic adjustment substances for osmotic adjustment in plants (Maet al., 2018).

Free proline was one of the components of plant proteins, and widely existed in plants in free form.Free proline could regulate the cell osmotic pressure, reduce cell acidity, regulate cell oxidation-reduction potential, eliminate ROS, and promote protein hydration (Anaet al., 2020).Therefore, if the plants were exposed to a low temperature, they accumulated free proline, which could significantly improve cold tolerance and plant protection (Zhang, 2019).With the treatment process, free proline content in Rivendel and in Haifa shoots first increased and then decreased and finally increased; and an earlier peak in Rivendel than that in Haifa indicated that Rivendel had a stronger chilling tolerance.The free proline content in shoots increased after 12 h, because the free proline synthesis mechanism was delayed under chilling stress.In general, the free proline content was low in plants.After low temperature stress, the free proline content could increase.Yanet al.(2019) found that rape had chilling tolerance because of the increase in free proline content under low temperature.

Soluble sugar was related to plant growth and development, yield formation and quality, plant respiratory metabolism and stress resistance.To adapt to low temperature, plants would actively accumulate soluble sugar to reduce the osmotic potential in cells, and the freezing point of cells would reach a low level to adapt to environmental conditions (Zhaoet al., 2019).In the present study, peak of soluble sugar content occurred more rapidly in shoots of Rivendel than that of Haifa under chilling stress, which meant Rivendel could respond to chilling stress more quickly than Haifa.Huet al.(2018) believed that there was a positive correlation in most plant cells between sugar content and cold resistance.Zhaoet al.(2020) found that the soluble sugar content showed alternating changes in researches on the effect of low temperature stress on amur grape.

Soluble protein level was very important under cold stress conditions, because it was useful in increasing cell osmotic pressure, which could protect the plant from damage caused by low temperature (Wuet al., 2019).In the present study, soluble protein content of plants was not constant, but changed upon exposure to lower temperatures.Soluble protein content in Rivendel and Haifa shoots first increased and then decreased and finally increased.Soluble protein content in Haifa shoots peaked earlier, but it was still lower than that in Rivendel at the same time, which indicated that Rivendel could endure a longer period of chilling stress than Haifa.Previous researches had found that soluble protein content was associated with enhancement of plant cold resistance.Liet al.(2018) found that low temperature stress could increase the content of soluble protein in white clover.Zhanget al.(2020) considered the soluble protein content in alfalfa to be significantly correlated with cold resistance.Tanet al.(2019) found that the soluble protein content of cold-tolerant cultivars was high in a study of cold resistance of Michelia leaves.

Conclusions

Rivendel and Haifa showed different physiological responses to chilling stress, which indicated that with the treatment process, Rivendel had a strong ability to maintain the stability of cells, cope with ROS and oxidative damage, and accumulate more osmotic adjustment substances compared with Haifa.The subordinate function value of Rivendel was higher than that of Haifa.These results showed that Rivendel had stronger chilling tolerance than Haifa.Comprehensive assessments on the physiological responses indicated that Rivendel had stronger chilling tolerance than Haifa, which could provide a reference value for the introduction of Rivendel and Haifa in northern China.