The physiological response of different tobacco varieties to chilling stress during the vigorous growing period

Tobacco is be sensitively affected by chilling injury in the vigorous growth period, which can easily lead to tobacco leaf browning during flue-curing and quality loss, however, the physiological response of tobacco in the prosperous period under low temperature stress is unclear. The physiological response parameters of two tobacco varieties to low temperature stress were determined. The main results were as follows: ① For tobacco in the vigorous growing period subjected to low-temperature stress at 4–16 °C, the tissue structure of chloroplast changed and photosynthetic pigments significantly decreased compared with each control with the increase of intensity of low-temperature stress. ② For tobacco in the vigorous growing period at 10–16 °C, antioxidant capacity of the protective enzyme system, osmotic adjustment capacity of the osmotic adjusting system and polyphenol metabolism in plants gradually increased due to induction of low temperature with the increase of intensity of low-temperature stress. ③ Under low-temperature stress at 4 °C, the protective enzyme system, osmotic adjusting system and polyphenol metabolism of the plants played an insignificant role in stress tolerance, which cannot be constantly enhanced based on low-temperature resistance at 10 °C. This study confirmed that under the temperature stress of 10–16 °C, the self-regulation ability of tobacco will be enhanced with the deepening of low temperature stress, but there is a critical temperature between 4 and 10 °C. The self-regulation ability of plants under low temperature stress will be inhibited.

Temperature is an important factor affecting the distribution of plant growth area, as well as the main environmental factor affecting the growth and development of plants 1,2 . The appropriate temperature is one of the basic conditions for plant growth and development. When the plant's environmental temperature is lower than the specific temperature range at which plants grow, the minimum value, low intensity, scope and duration changes would lead to plant adaptive response. At the same time, the low temperature could make plant grow slowly, tissues and organs lose water and wilt, leaf turn yellow and show water stains, susceptible to diseases and insect pests, production decline in the quality and even plant die 3,4 . As a leaf cash crop widely cultivated, chilling injury is also a common natural disaster on tobacco production. According to the relevant research reports, in Yunnan, the largest growing area in China, low-temperature chilling injury causes serious damage to the tobacco growers. In the subsequent tobacco flue-curing process, it can also increases the brown reaction, which leads to a significant reduction in the apparent quality and taste, and causes huge economic losses 5,6 . Therefore, it has become the flash point and focus of tobacco abiotic stress research to study the physiological response mechanism of tobacco to low temperature stress, so as to reduce yield loss caused by cold injury.
At present, a large number of studies have been carried out around the world on the response of tobacco plant to low temperature stress. Zhou et al. studied the metabolism of polyphenols in tobacco seedlings under low temperature stress. It was found that low temperature stress could promote the synthesis of lignin in polyphenol metabolism of tobacco seedlings and enhance the protective effect of cell wall by increasing the content of lignin 7  and SPSS18.0 software and Duncan's multiple range test was carried out. The data in this study was presented as the mean of three replicates and the significant level was set at alpha level (P < 0.05). The physiological indexes were comprehensively analyzed by utilizing the membership function method. When physiological indexes are positively correlated with stress effects, the membership function is shown as follows: If physiological indexes have a negative correlation with stress effects, the membership function is expressed as follows: where, U max and U min indicate the maximum and minimum values of a series of indexes, respectively; U x represents the measured values of corresponding series of indexes. Finally, the weighted averages of membership function values of each index are calculated.
Experimental material statement. The use of plants parts in the present study complies with international guidelines.

Results and analysis
Changes of chloroplast ultrastructure under different low-temperature stresses. As shown in Figs. 1 and 2, at 25 °C, chloroplasts of tobacco leaves, which are oval or rhombic, were tightly attached onto the cell wall. The long axis was parallel to the cell wall, and starch granules were few and completely wrapped in chloroplasts. Moreover, grana lamellae were closely arranged, on which a small number of osmiophilic granules were attached. Compared with 25 °C, with increasing duration of low-temperature stress, the structure was destroyed and the membrane system was gradually broken. Starch granules in chloroplasts enlarged, and more osmiophilic granules appeared. Moreover, thylakoid lamellae became loose and distorted. For the same variety, the lower the temperature was, the more obviously the chloroplast structure changed. Under 16 °C treatment, Hongda variety with strong resistance on day 1 had no obvious difference with 25 °C. On day 5, the volume of starch granules significantly expended and a few osmiophilic granules appeared. Under 10 °C and 4 °C treatments, chloroplast structures in tobacco leaves changed obviously as early as day 1. On day 5, deformation degree of chloroplasts, volume of starch granules, degree of expansion and disintegration of thylakoids and number of osmiophilic granules reached the maximum. Under 16 °C treatment, K326 variety had a certain difference in chloroplast structure on day 1 compared with 25 °C, mainly shown as enlarged starch granules. On day 5 under 10 °C and 4 °C treatments, the volume of starch granules expanded obviously and a small number of osmiophilic granules were observed. Under 10 °C and 4 °C treatments, the change laws of chloroplast structures of K326 variety were same with those of Hongda variety. By comprehensively analyzing changes of chloroplast ultrastructures of the two tobacco varieties under low-temperature treatment, it is found that the chloroplast structure of K326 variety changed greater than that of Hongda variety under the same treatment.
Influences of low-temperature stress on REC of tobacco leaves. As demonstrated in Fig. 3, under 16 °C and 10 °C treatments, RECs of tobacco leaves of Hongda and K326 varieties firstly rose and then reduced with prolonging low-temperature stress, while they constantly increased with the increasing duration of exposure to stress under 4 °C treatment. Under the same low-temperature treatment, REC of leaves of K326 variety with weak low-temperature resistance rose largely compared with Hongda variety with strong low-temperature resistance. However, for the same variety, the lower the temperature, the faster the REC rose and the larger the Impacts of different low-temperature stresses on content of osmotic adjusting substances in tobacco leaves. Effects of different low-temperature stresses on SS content in tobacco leaves. As displayed in   Influences of low-temperature stress on SP content in tobacco leaves. Figure 6 illustrated that under 16 Figure 9 showed that chlorophyll a + b content in leaves of Hongda and K326 varieties both decreased with the increase of days of exposure to lowtemperature stress under the three treatments. Under the same low-temperature stress, chlorophyll a + b content in leaves of K326 variety with weak low-temperature resistance reduced faster than that in Hongda with strong low-temperature resistance. As for the same variety, the lower the temperature, the faster the chlorophyll a + b content decreased. Effects of low-temperature stress on antioxidant enzyme system in tobacco leaves. Influences of different low-temperature stresses on SOD activity in tobacco leaves. As shown in Fig. 10, under 16 °C treatment, SOD activity in leaves of Hongda variety rose and tended to be stable with prolonging duration of low-temperature stress. SOD activity in 1-5 days significantly increased compared with 25 °C and reached the highest level in 3-5 days. Under 10 °C and 4 °C treatments, SOD activities in leaves firstly rose and then reduced with the increase of days under low-temperature stress based on the fact that they were remarkably higher than those in 25 °C. Under the two treatments, SOD activities separately reached the highest level in 2-4 days and    For the two tobacco varieties, low-temperature treatment could improve CAT activity in tobacco leaves. Under the same low-temperature treatments, the increase amplitude of CAT activity in leaves of the low-temperature resistant variety (Hongda) was greater than that of the low-temperature susceptible one (K326). For the same variety, the comparison of the maximum increase amplitudes of CAT activities under each treatment showed that the amplitudes were ranked in a descending order under 10 °C, 4 °C and 16 °C treatments. Moreover, the maximum CAT activity was inhibited under 4 °C treatment.

Changes of chlorophyll a + b content under different low-temperature stresses.
Influences of low-temperature stress on polyphenol metabolism in tobacco leaves. Effects of low-temperature stress on PPO activity in tobacco leaves. Figure 13 illustrated that under 16 °C treatment, PPO activity in leaves of Hongda variety firstly increased and then tended to be stable with the increasing days under stress and reaches the maximum on day 3, while there was no significant difference in changes of PPO activity in 3-5 days. Under 10 °C and 4 °C treatments, PPO activities in leaves of Hongda variety were both significantly higher than those under 25 °C and they first increased and then decreased with the rise of days of exposure to stress. The activity reached the maximum on day 3 under 16 °C treatment and maintained at the highest level in 3-4 days under 4 °C treatment. Under 16 °C treatment, PPO activity in leaves of K326 variety showed the same change trend with Hongda variety, that is, it tended to be stable after rising. The activity reached the maximum after 2 d and then stabilized at this level. Under 10 °C treatment, PPO activity in leaves of K326 variety firstly   Fig. 14 Influences of low-temperature stress on total phenol content in tobacco leaves. As demonstrated in Fig. 15, under 16 °C treatment, as low-temperature stress duration prolongs, accumulation of phenolic substances in leaves of Hongda variety first rose and then is inclined to be stable, and the highest content was found in 4-5 days. Under 10 °C and 4 °C treatments, the content of phenolic substances firstly increased and then decreased with stress duration. In different periods, the content of phenolic substances rose compared with those in 25 °C and reached the highest level on day 3. In each period under 16 °C and 10 °C treatments, the content of phenolic substances  www.nature.com/scientificreports/ accumulated in leaves of K326 variety increased compared with those in 25 °C and they rose and tended to stabilize with prolonging low-temperature stress duration. The highest contents under the two treatments were found on days 4 and 3, respectively. Under 4 °C treatment, the content of phenolic substances in leaves of K326 variety significantly increased compared with that on day 0. In addition, with the increase of days of exposure to stress, the content firstly rose and then reduced, and it reached the highest level on day 3 and then declined. The results showed that for the same variety under different treatments, when the content of phenolic substances in leaves reached the highest level, the 10 °C, 4 °C and 16 °C were ranked in a descending order by the increase amplitudes of the content. In a certain temperature range, with the decrease of temperature, phenolic substances accumulated in leaves increased fast and their content rose. Under 4 °C treatment, as the strongest stress treatment in this experiment, the capacity of accumulating phenolic substances was lower than that under 10 °C treatment. Table 1, through use of the membership function, a mathematical tool characterizing a fuzzy set, cold resistance of Hongda and K326 varieties were comprehensively evaluated based on ten measured indexes. By taking the average of membership degrees of the ten indexes as the comprehensive identification standard for cold resistance under such treatments, the larger the average was, the stronger the cold resistance, and vice versa. Data on day 5 under low-temperature stresses were selected for solving the membership function. The results showed that the average membership degrees of cold resistance indexes of Hongda variety under 16 °C, 10 °C and 4 °C treatments were 0.05, 0.83 and 0.12, respectively. Over 5 d of different low-temperature treatments, cold resistance of plants under 10 °C treatment was the strongest, followed by that under 16 °C treatment, while the weakest cold resistance under 4 °C treatment. The average membership degrees of cold resistance indexes of K326 variety under 16 °C, 10 °C and 4 °C treatments separately were 0.56, 0.75 and 0.16 and cold resistance under different low-temperature treatments were ranked in the same order with that of Hongda variety. The comprehensive results showed that tobacco still maintained under strong low-temperature resistance from 16 to 10 °C treatments for 5 days. The low-temperature resistance rose with the increase of intensity of low-temperature stress. Under aggravated stress, plants could improve their low-temperature resistance through self-regulation of metabolism, which was beneficial to reducing the damages of low temperature to plants. However, the weakest resistance of plants was shown under 4 °C treatment. In this case, the metabolism in plants may be seriously damaged by low temperature, and the reduction of low-temperature resistance would seriously damage plants under severe low-temperature stress.

Changes of chloroplast ultrastructure under different low-temperature stresses.
Chloroplasts are a kind of organelles which are susceptible to environmental changes. Their structural changes, the degree of thylakoid stacking, and the numbers of starch granules and osmiophilic granules change adaptively with the environment 19,20 . It is generally believed that Low temperature inhibits the synthesis and transportation of assimilation products. Products are rapidly transformed into starch and accumulated into large starch grains in chlorophylls 21 . The increase of starch grains is an adaptation of plants to low temperature environment, and osmiophilic granules are the result of degradation of thylakoid membrane of chloroplasts 22,23 . By observing tobacco seedlings with a SEM, Li et al. found that low-temperature stress can lead to deformation of chloroplasts, loose arrangement of grana lamellae, enlarging volume of starch granules and increasing number of osmiophilic granules, which is also confirmed in this study 8 . When the two tobacco varieties were treated at 16, 10 and 4 °C, osmiophilic granules appear and become increasingly more with prolonging stress time. Furthermore, the number of osmiophilic granules in K326 variety under each treatment was greater than that in Hongda variety, indicating that chloroplasts in tobacco leaves of the variety with good cold resistance confer better structural stability. Therefore, the adaptation of chloroplast structure to low temperature environment can be used as one of the criteria to judge the cold resistance of tobacco.

Changes of photosynthetic pigment under different low-temperature stresses.
Chloroplast pigment plays an important role in the growth and modulation of flue-cured tobacco 24,25 . In this experiment, chlorophyll a, chlorophyll b and total chlorophyll content in Hongda and K326 varieties under low-temperature www.nature.com/scientificreports/ treatments significantly reduced compared with those in 25 °C. With the increase of days of exposure to lowtemperature stress, they constantly decreased with an increasingly large amplitude. For the same tobacco variety, chlorophyll a, chlorophyll b and total chlorophyll content under low-temperature treatments change consistently, that is, the lower the temperature is, the faster the decrease and the larger the decrease amplitude, which is consistent with the research results obtained by Liang et al. 26 . By comparing the two tobacco varieties under the same low-temperature treatments, it is found that chlorophyll a, chlorophyll b and total chlorophyll contentsin Hongda variety reduce slower with a smaller amplitude compared with those in K326 variety, which coincides with the research results of Chen et al. 27 . It also explain that the low temperature will break the balance of chlorophyll synthesis and degradation of the original, and result in changes of chlorophyll content. The strong low temperature resistant variety (HongDa) photosynthetic pigment drops less than the variety (K326) of which resistance is weak. On the one hand, it may be due to the weak chloroplast structure stability of the less resistant variety. At the same time, chloroplast synthase activity decreased significantly, and the low temperature caused the disorder of chloroplast function. On the other hand, it may be due to the oxidation rate of PSII electron transport primary quinone receptor QA is more restricted, and the photochemical quantum efficiency of PSII reaction center recovered more slowly 28 .

Changes of REC and MDA content under different low-temperature stresses. REC is an index
to measure the degree of electrolyte leakage in cells, and an important reference index for damage degree of plant cell membrane 29,30 . Qi et al. found that low-temperature stress can lead to disorder of plant cell membrane system and REC is positively correlated with the intensity of low-temperature stress, which is confirmed in this research 31 . Under the same treatment, the REC of K326 variety increased with larger amplitude than that of Hongda variety. RECs of the two varieties under the low-temperature treatment at 4 °C constantly increased, and when RECs reach the peak under each treatment, the increase amplitudes under 4 °C, 10 °C and 16 °C treatments were ranked in a descending order. As one of the final products of membrane lipid peroxidation, MDA can also effectively reflect the damage degree of cell membrane 32,33 . The changes of MDA content in leaves of Hongda and K326 varieties under each treatment showed that for the same variety, the lower the temperature is, the higher the peak MDA content, which causes more serious damages to plants. Under the low-temperature stresses at 16 °C and 10 °C, plants can effectively reduce damages caused by low temperature in a short time through self-regulation of metabolism. However, under the low-temperature stress at 4 °C, the damages were not reduced in 5 days, which was consistent with the research results obtained by Wang Jiachuan concerning changes of relevant enzymes in different tobacco varieties induced by low-temperature 34 . This may be due to the damage of membrane lipids caused by reactive oxygen species under low temperature stress of 4 °C, which leads to the damage of cell function. It is consistent with the research results of Wang Jianchuan on the changes of related enzymes in different flue-cured tobacco varieties under low temperature induction. Under the same low temperature stress treatment, the increase of MDA content in leaves of K326 with weak low temperature resistance is greater than that of HongDa with strong low temperature resistance, which may be because the varieties with weak resistance are more sensitive to low temperature stress. Low temperature leads to the rapid increase of intracellular reactive oxygen species, which increases the degree of membrane lipid peroxidation and eventually produces a large amount of membrane peroxidation product MDA.
Changes of content of osmotic adjusting substances under different low-temperature stresses. Zhang et al. found that SS and SP are important osmotic adjusting substances in plants and the increase of their content is positively correlated with cold resistance of plants 35 . The results showed that lowtemperature stress rises SS content in tobacco leaves. Both Hongda and K326 varieties under 16 °C and 10 °C treatments showed that the capacity of accumulating SS in plants rose with the increase of intensity of low-temperature stress. However, under 4 °C treatment, the capacities of plants of the two tobacco varieties accumulating SS and SP were weakened compared with that under 10 °C treatment, especially K326 variety, with capacity to accumulate SS and SP even lower than that under 16 °C treatment. This indicates that under the treatment at 4 °C, the capacity of tobacco to enhance low-temperature resistance by regulating content of osmotic adjusting substances has decreased. At present, some reportse have pointed out that genes and functional proteins are involved in plant low temperature stress 36 , but the research is not in-depth enough. In the future, we should explore the key genes that induce cold domestication and cold tolerance of tobacco 37 , especially the genes related to SS metabolism, and make use of modern biotechnology methods to improve the cold resistance of tobacco, so as to provide the basis for the breeding of good varieties and new varieties of tobacco germplasm resources with strong cold tolerance, and expand the cultivation range of tobacco.
Changes of protective enzyme activity under different low-temperature stresses. SOD is one of the protective enzymes in plants 38 . It has been found that the resistance of plants is related to the maintenance of high SOD level in plants under stress 39 . The research results demonstrate that SOD activities of the two tobacco varieties both increase significantly under low-temperature treatment, and the increase amplitude of low-temperature tolerant variety is greater than that of low-temperature susceptible variety. Ma et al. also found that in a certain range of low-temperature intensity, SOD activity increases with the decrease of temperature 40 . However, when the temperature exceeds a certain critical value, its activity will decrease with intensifying stress, which is also confirmed in this study. Under 16 °C treatment, SOD activities in leaves of the two tobacco varieties both firstly rose and then tend to be steady. Under 10 °C and 4 °C treatments, SOD activities in leaves of the two varieties increased in the early stage and decreased in the later stage of low-temperature stress. www.nature.com/scientificreports/ POD and CAT, as important enzymes to remove H 2 O 2 in plants, play an important role in alleviating peroxidative damages and enhancing low-temperature resistance of plants under stress [41][42][43] . In this study, POD and CAT activities in leaves of the two tobacco varieties induced by low-temperature treatments both can significantly rise compared with those in 25 °C. Under 16 °C treatment, POD and CAT activities in leaves of K326 variety firstly increased and then tended to be stable with the increase of stress duration. Under 10 °C and 4 °C treatments, POD and CAT activities increased in the early stage and decreased in the later stage. For Hongda variety, under each low-temperature treatment, POD and CAT activities firstly rose and then declined with increasing stress duration, indicating that difference in cold resistance of the varieties was related to POD and CAT contents in leaves. For the same variety, the maximum increase amplitudes of POD and CAT activities were ranked in a descending order under 10 °C, 4 °C and 16 °C treatments. In other words, in a certain range of low-temperature stress, the lower temperature can stimulate higher POD and CAT activities in plants. However, when the temperature reaches 4 °C, the maximum enzyme activity is inhibited, which was consistent with the conclusion of Zhang et al. 44 . the reason may be in a certain range of chilling stress induced signal mechanism and cold stress response gene expression of tobacco, activate the protective enzyme system 45,46 , effectively tolerate freezing stress, while with the aggravation of stress, the production of H 2 O 2 in plants increases, resulting in the decrease of POD and CAT scavenging efficiency. In the end, the damage to the plant caused by excessive H 2 O 2 was aggravated.

Changes of polyphenol metabolism under different low-temperature stresses. PPO and PAL
are important enzymes in polyphenol metabolism of plants, which can effectively improve resistance of plants to diseases and insect pests and play an important role in resistance to external stress, and in photosynthesis and biosynthesis [47][48][49] . Under stress, high PPO and PAL activities are conducive to enhancing stress resistance of plants. This research showed that compared with 25 °C, the increase amplitudes of the highest levels of PPO and PAL activities in leaves of the same variety were ranked in a descending order under 10 °C, 4 °C and 16 °C treatments. Low temperature results in higher PPO and PAL activities. In addition, for different varieties under the same treatment, PPO and PAL activities in leaves of Hongda variety increased faster, with larger amplitude. This may be due to the activation of the defense system, especially the activation of phenylpropane metabolism, or the up-regulated expression of the upstream gene PAL, which promotes the increase of PAL activity to produce more plant protectants and lignin to alleviate the damage. These potential physiological responses can be used in the future molecular level study of tobacco cold resistance. Previous studies demonstrate that with the increase of intensity of low-temperature stress, PPO and PAL activities can be gradually enhanced 50 . However, in this experiment, the activities under 4 °C treatment were lower than those under 10 °C treatment, suggesting that extremely low temperature inhibits PPO and PAL activities to some extent.
Some studies showed that the more intensified low-temperature stress can stimulate plants to accumulate more phenolic substances to reduce damages of free radicals to plants 51 . This study illustrated that when the content of phenolic substances in leaves of the same variety under different treatments reached the highest level, the increase amplitudes were ranked in a descending order under 10 °C, 4 °C and 16 °C treatments. In a certain temperature range, with the decrease of temperature, the accumulation rate and content of phenolic substances in leaves increased. Under 4 °C treatment, as the strongest stress treatment in this experiment, the accumulation capacity of phenolic substances was lower than that under 10 °C treatment. The reason may be that under the treatment at 10-16 °C, tobacco plants were stimulated to accumulate a large amount of phenolic substances to reduce the damage of free radicals. Under the treatment at 4 °C, plants suffered from high-intensity stress, while their capacity to accumulate phenolic substances reduces. The pathway of removing lots of ROS produced under stress by using phenolic substances as non-enzymatic antioxidants in plants is blocked, so the low-temperature resistance of plants is relatively reduced.

Conclusions
The change laws of Hongda and K326 varieties under the three low-temperature stresses were comprehensively analyzed. Based on this, it is found that for tobacco in the vigorous growing period under low-temperature stress at 10-16 °C, with increasing intensity of stress, antioxidant capacity of protective enzyme system, osmotic adjustment capacity of osmotic adjusting system and polyphenol metabolism are all enhanced by low-temperature stress. Such changes are conducive to timely improving the low-temperature resistance of plants, in order to survive under low-temperature stress. When temperature decreases to 4 °C, the protective enzyme system, osmotic adjusting system and polyphenol metabolism of plants fail to make a full function in stress resistance, which cannot be continuously enhanced on the basis of resistance at 10 °C, while instead inhibited to some extent. Two tobacco varieties, especially K326, are damaged seriously when they are exposed to 4 °C for a long time. The results of changes of chlorophyll, REC and MDA content and membership function of cold resistance indexes under different treatments prove the above conclusions. Therefore, it is considered that there is a critical temperature between 4 and 10 °C for tobacco in the vigorous growing period. When the temperature was lower than the critical value, the self-regulation capacity of tobacco plants under low-temperature stress begins to be inhibited.

Data availability
The data presented in this study is contained within the article.