The Effects of the Field Cold Stress in the Harvest Period on the Quality and Curing Characteristics of Tobacco Leaves


 Background: Climate change in high-altitude areas causes tobacco (Nicotiana tabacum L.) in its mature period to be subjected to cold stress, which damages the yield and quality of tobacco leaves. We found that abrupt diurnal temperature difference (the daily temperature drops more than 20℃), along with rainfall occurred in the tobacco-growing areas at an altitude above 2450 m, which caused cold stress to tobacco in field. Results: The change process of the surface color of tobacco leaves was normal to dark green (within 1 to 2 d), to purple red (within 2 to 3 d), then dark red (within 3 to 4 d), and finally off-white (within 4 to 6 d), the final leaves appear obvious large areas browning, and the curing availability was extremely poor. Further research found the quality of fresh tobacco leaves, the content of key chemical components, and the quality of production are greatly reduced by cold stress. The reason was cold stress in high altitude environment destroyed the antioxidant enzyme system of mature flue-cured tobacco. Cold stress in high altitude environment destroyed the antioxidant enzyme system of mature flue-cured tobacco. The quality of fresh tobacco leaves, the content of key chemical components, and the quality of production are greatly reduced by cold stress.Conclusion: In conclusion, this study confirmed that the field cold stress in high-altitude tobacco areas was the main reason for the obvious browning tobacco leaves during the tobacco curing process. This adversity environment seriously damaged the quality of tobacco leaves.

of superoxide dismutase (SOD) and catalase (CAT) are signi cantly lowered. Under the synergistic effect, the active oxygen in tobacco is eliminated so it can maintain at a low level, thus reducing the damage to plants [19][20].
Laojun Mountain Town is located in the northwest of Jianchuan County, Dali Bai Autonomous Prefecture of Yunnan Province in a high-altitude intermountain basin, with an average altitude of 2450 m, an annual average air temperature of 10 °C, annual precipitation of 960 mm, a sunshine duration of 2200 h, a frost duration of 210 to 220 d, a heavy frost period of about 90 d, and frequent hail in summer and autumn. Tobacco is one of the major economic crops grown on Laojun Mountain, in which Honghuadajinyuan (HD) is the main variety, showing favorable cold hardiness. According to recent data from 2019, the planting area of tobacco in the town has reached 173.2 ha − 1 , with a yield of 1950 to 2250 kg ha − 1 and an average price of 4.93dollar kg − 1 . Moreover, the area subjected to the eld cold stress is about 64 ha − 1 , which accounts for 36.95% of the total planting area. During eld cold stress, some middle leaves and the majority of the upper leaves of tobacco cannot be cured, which results in the direct economic loss of 184,000 dollars to local tobacco growers.
Existing reports concentrate on exploring the changes of the appearances and physiological and biochemical indices of tobacco seedlings in the environment of arti cial cold stress, however, related research into eld cold stress on tobacco in the harvest period remains rare so it is important to investigate the mechanism underlying the eld cold stress of tobacco generated based on the unique natural climate conditions of Yunnan Province. The purpose of the study is to enrich the mechanism underlying the eld cold stress on tobacco on natural conditions of Yunnan Province. It is expected to provide a certain theoretical basis for inducing factors and mechanism underlying the eld cold stress on tobacco in the harvest period on natural conditions and feasible methods of reducing the economic loss suffered by tobacco growers.

Changes in Meteorological Factors Generating Field Cold Stress
It can be seen from Fig. 1 that the experimental base located in Jianji village of Laojun Mountain Town showed signi cant uctuations in temperature and rainfall in August: the daily highest and lowest temperature varied in the ranges of 21.5 to 41.9 °C and 9.4 to 17.1 °C, respectively, with the temperature difference in the range of 12.1 to 24.6 °C; the daily precipitation varied within 0 to 21.6 mm. The maximum diurnal temperature difference (30.1 °C) was recorded on 12 August; however, no rainfall occurred thereon; the minimum diurnal temperature difference (8.7 °C) was found on 1 August, accompanying with 5.9 mm of precipitation. According to the survey results of local eld cold stress, it can be found that substantial cooling accompanying meteorological disasters (including rainfall and a small amount of hail) appeared on 16 and 17 August; in the next six days, tobacco showed a range of symptoms caused by cold stress (Fig. 2). The tobacco leaves in the middle and upper parts suffered the most serious damage. The surface color of the tobacco leaves changed from normal to dark green (within 1 to 2 d), to purple red (within 2 to 3 d), then dark red (within 3 to 4 d), and nally off-white (within 4 to 6 d). Eventually, a large area of scalded leaves was found, showing poor ue-curing availability.
To explore the speci c causes for cold stress, the meteorological data collected inside and outside the greenhouse on 16 to 17 August were analyzed (Fig. 3).
It can be found from Fig. 3 that the temperature outside the greenhouse (natural conditions) varied within 9.4 to 34.8 °C, with a difference of 25.4 °C; the temperature inside the greenhouse varied between 12.5 to 36.2 °C, with a difference of 23.7 °C. The rainfall occurring in the cooling period was concentrated between 18:00 to 22:00 on 16 August, with a total precipitation of 5.2 mm. The tobacco leaves inside and outside the greenhouse in the experimental base were sampled on 19 August (Fig. 4). The 10th leaf (counted from the bottom to up) was sampled. It can be seen from Fig. 4 that the tobacco leaves inside the greenhouse showed no signi cant symptoms caused by cold stress, except for having a small number of brown spots on leafstalk; tobacco leaves outside the greenhouse exhibited signi cant red and puce plaques from the middle part of leaves to the leafstalk and the leaves turned from green to yellow on the whole.
It was judged that tobacco leaves outside the greenhouse were subjected to cold stress.
Comparison of Slices of Tobacco Leaves Inside and Outside the Greenhouse As shown in Fig. 5 and Table 1, the thicknesses of laminae, palisade tissues, and spongy tissues and the ratio of the thickness of palisade tissues to that of spongy tissues (tissue ratio) of fresh tobacco leaves from inside the greenhouse showed a signi cant difference (P < 0.05) from those outside. Table 1, the thickness of the laminae, palisade tissues, spongy tissues, upper epidermis and lower epidermis of fresh tobacco leaves outside the greenhouse was lower than that inside the greenhouse, the values inside the greenhouse were separately 151.76%, 105.19%, 175.90%, 91.97% and 222.35% greater than those outside, which showed a signi cant difference. On the contrary, in terms of the ratio of the thickness of palisade tissues to that of spongy tissues (tissue ratio) of fresh tobacco leaves, the value outside the greenhouse was 25.27% greater than that inside the greenhouse. Comparison of Water Loss Rates of Tobacco Leaves Inside and Outside the Greenhouse in the Flue-curing Process

As shown in
As shown in Fig. 6 and Fig. 7, under different curing stages, there were signi cant differences (P < 0.05) in water loss rate and moisture content of tobacco leaves; under the same curing stage, only at 48 °C showed a signi cant difference (P < 0.05) in moisture content of tobacco leaves inside and outside the greenhouse.
With the increase of the ue-curing time, the moisture contents and water loss rates of tobacco leaves inside and outside the greenhouse slowly changed at rst, then rapidly varied and nally changed slowly again. The moisture content of tobacco leaves inside the greenhouse was generally higher than that outside, while the water loss rate of the former was lower than that of the latter. In terms of the moisture content, the changes in the moisture content of the tobacco leaves in treatments inside and outside the greenhouse from 25 °C (fresh tobacco leaves) to 54 °C were 83.28 to 24.95% and 78.42 to 12.72%, respectively. The moisture content was greatly changed in the ue-curing process at 42 to 54 °C: the moisture contents of tobacco leaves inside and outside the greenhouse at 42 °C were separately 177% and 362% higher than those at 54 °C; the moisture contents of tobacco leaves in the curing process at 42 °C, 48 °C, and 54 °C inside the greenhouse were separately 17.80%, 123.19%, and 96.15% higher than those outside.
As for the water loss rate, its change amplitudes for tobacco leaves inside and outside the greenhouse at 38 to 54 °C were 16.14 to 92.66% and 35.3 to 97.3%, respectively; the water loss rates of tobacco leaves inside and outside the greenhouse at 48 °C were separately 382% and 161% higher than those at 38 °C; the water loss rates of tobacco leaves outside the greenhouse in the curing process at 38 °C, 42 °C, and 48 °C were separately 118.71%, 27.86%, and 18.26% higher than those inside the greenhouse.
Comparison of SPAD Values and Plamochromic Pigments in Tobacco Leaves Inside and Outside the Greenhouse During Flue-curing As shown in Table 2, the SPAD values and plamochromic pigments in tobacco leaves inside and outside the greenhouse in different ue-curing stages both presented a signi cant difference (P < 0.05).
In the ue-curing process, the values of the SPAD, chlorophyll a, and chlorophyll b in tobacco leaves inside the greenhouse were all greater than those outside the greenhouse. The three indices of tobacco leaves inside and outside the greenhouse tended to decrease rapidly at rst and then slowly decreased. They were degraded at the fastest rate at 38 °C and then the rate of degradation decreased rapidly, then tended to stabilize. The SPAD values of tobacco leaves inside the greenhouse were 29.79 to 172.57% greater than those outside the greenhouse, and were the lowest and the highest in fresh tobacco leaves and in leaves ue-cured at 38 °C, respectively; the values of the chlorophyll a in tobacco leaves inside the greenhouse were 148.94 to 1153.91% greater than those outside the greenhouse, in which the lowest and the highest values were found in fresh tobacco leaves and in leaves ue-cured at 42 °C, respectively; the values of the chlorophyll b content in tobacco leaves inside the greenhouse were 73.91 to 628.26% larger than those outside the greenhouse, with the lowest and the highest values at 52 °C and 42 °C, respectively.
The values of the lutein and β-carotene content in tobacco leaves inside the greenhouse were all greater than those outside. Under ue-curing, the lutein and βcarotene contents in tobacco leaves inside and outside the greenhouse rose at rst and then decreased. The two indices inside the greenhouse reached their maxima separately at 42 °C and 48 °C while the outside of were lutein and β-carotene content maximized at 54 °C. The chlorophyll contents of tobacco leaves inside and outside the greenhouse separately varied between 150.30 to 297.14 µg g − 1 and 93.28 to 139.42 µg g − 1 , in which the value inside the greenhouse was 48.21 to 164.57% greater than that outside. Moreover, the index was separately minimized and maximized at 54 °C and 42 °C. The contents of β-carotene in tobacco leaves inside and outside the greenhouse separately varied between 1940.38 and 4489.59 µg g − 1 and 1207.53 to 1888.94 µg g − 1 . The content of βcarotene in tobacco leaves inside the greenhouse was 60.69 to 149.34% greater than that outside. The values were separately maximized and minimized in fresh tobacco leaves and in ue-cured leaves at 48 °C.

Comparison of Chemical Compositions and Polyphenols in Tobacco Leaves Inside and Outside the Greenhouse During Curing
It can be seen from Table 3 that conventional chemical composition indexes and polyphenols all presented signi cant differences inside and outside the greenhouse.
With increased ue-curing time, the starch contents of tobacco leaves inside and outside the greenhouse gradually decreased, exhibiting the fastest reduction in fresh tobacco leaves at 38 °C; the total sugars, reducing sugars, and sugar-nicotine ratio increased at rst, then decreased, reaching a peak at 38 °C. Various indices used to classify tobacco leaves were greater outside the greenhouse than inside. The starch contents of tobacco leaves inside and outside the greenhouse separately varied within 1.04 to 33% and 5.4 to 39.94%, in which the index value of tobacco leaves outside the greenhouse was 21.03 to 567.72% greater than that inside. The minimum and the maximum values separately appeared in fresh tobacco leaves and initially ue-cured tobacco leaves. The analyses of the total sugars, reducing sugars, and sugar-nicotine ratio were similar to that for starches.
The chlorogenic acid and rutin content in tobacco leaves inside and outside the greenhouse gradually increased, as described above. As can be seen from Fig. 8, under the activities of SOD, POD and CAT, the activity of the three enzymes in the greenhouse was higher than that outside the greenhouse, and showed a trend of increase at rst and then decreased. The activity of the three enzymes was the highest at the stage of curing at 38 °C to 42 °C, and then decreased sharply. Meanwhile, under the same curing time, the SOD, POD and CAT enzymes treated in the greenhouse could maintain high activity more effectively than those treated outside the greenhouse. The content of MDA in the treatment inside and outside the greenhouse increased with the progress of curing, and the treatment outside the greenhouse increased sharply after 42 °C and exceeded that of the treatment inside the greenhouse at 48 °C.
The PPO activity of both inside and outside the greenhouse treatment showed a trend of a small decrease, then a sharp increase and then a sharp decrease, the indoor treatment increased abruptly at 38 to 42 °C, then decreased sharply, while the out-of-greenhouse treatment increased abruptly at 42 to 48 °C, and then decreased sharply.

Comparison of Economic Traits and Sensory Evaluation of Initially Flue-cured Tobacco Leaves Inside and Outside the Greenhouse
It can be seen from Fig. 9 that there is an obvious difference in the appearance of the rst-cured tobacco leaves inside and outside the greenhouse. There is bright color, good opening, no obvious miscellaneous color and hanging ash inside the greenhouse. However, outside the greenhouse, tobacco leaves have gray and dark color, small opening, obvious miscellaneous color and hanging ash on the surface.
As can be seen from Table 4, there are signi cant differences in the output, output value and average price of tobacco leaves inside and outside the greenhouse. Each index shows that inside the greenhouse is higher than that outside the greenhouse, and the yield inside the greenhouse is 13.45%, 47.32%, 37.32% and 29.85% higher than that outside the greenhouse, respectively. As can be seen from Table 5, the total score of sensory quality of tobacco leaves in the greenhouse was signi cantly higher than that of tobacco leaves outside the greenhouse by 20.44%.   The different capital letters indicate signi cant between different treatments at the same stage (P < 0.05). Values represent the averages of three biological replicates.

Discussion Ecological Factors behind the Field Cold Stress in the Harvest Period of Tobacco in Yunnan Province
Altitude, temperature, and rainfall are the major ecological factors inducing the eld cold stress in the harvest period of tobacco [21]. The tobacco in Yunnan Province was affected by low temperature in the planting and harvest periods while insu cient heat affected the middle growth period of tobacco leaves. The altitudes of the tobacco-growing areas were mainly between 1300 and 2000 m; the change amplitude of the temperature in the harvest period was between 17 and 22 °C; the precipitation in August and September was between 95 to 180 mm [22]; however, in the tobacco-growing areas at altitudes higher than 2000 m, the solar radiation increased; the effective accumulative temperature decreased; the diurnal temperature difference rose. The killing cold stress frequently appeared in the harvest period of tobacco. The experimental site was located at a measured altitude of 2565 m, which was much higher than that of the main tobacco-growing areas in Yunnan Province. The monthly temperature in the harvest period changed signi cantly: when the change in air temperatures in August was between 9.4 and 41.9 °C, this was extremely likely to trigger in situ cold stress. Through investigation, it was found that the temperature dropped by 25.4 °C at the experimental site on the day subjected to the cold stress. Within the subsequent six days, the appearance and color of middle and upper tobacco leaves changed suddenly, turning from green to off-white, which was related to the results of existing research that suggest that cold stress can damage the photosynthetic system of plant leaves to reduce the content of chloroplast pigments [23]. Additionally, the high altitude also led to the signi cant change of the light intensity and light quality, thus increasing the ultraviolet irradiation, which was probably one of reasons why the photosynthetic system was damaged after cold stress [24]. Under low-temperature climate conditions, rainfall readily caused damage such as freezing-injury and hail impact. A small amount of rainfall was detected, accompanying with a natural disaster, hail, on the day subjected to cold stress in the experimental spot, which was also one of reasons directly causing cold stress to tobacco.

The Effects of the Field Cold Stress on the Quality and Curing Characteristics of Fresh Leaves of Tobacco in the Harvest Period
Cold stress can trigger the thickening, wilting due to water loss, and destruction of the photosynthetic system of tobacco leaves [25]. The quality of fresh tobacco leaves is considered to be the primary factor determining the curing characteristics and even the yield and quality of tobacco leaves, which is shown in various aspects such as microstructure, moisture, pigment, chemical composition, and enzyme activity [26] (Zhang et al., 2018). The research results showed that the thickness of lamina, lamina, upper epidermis and lower epidermis, palisade tissue and sponge tissue of tobacco leaves (with cold damage) outside the greenhouse was signi cantly lower than that of fresh tobacco leaves (without cold damage) in greenhouse, indicating that the cold stress destroyed the tissue structure of leaves and led to the weakening of leaf assimilation ability, which was consistent with the results of Zhang 2014 [27].
However, in the results of this study, the ratio of thickness of palisade tissues to that of spongy tissues (tissue ratio) in the greenhouse was lower than that outside the greenhouse, which was contrary to the results of Zhang 2014 [27], which may be due to the structural changes of palisade tissue and sponge tissue induced by chilling stress, so as to improve the resistance to stress. The SPAD value and the content of chloroplast pigments of fresh tobacco leaves outside the greenhouse were both lower than those inside, indicating that chilling stress had a great effect on the Plastid pigment of tobacco leaves, which would seriously reduce the content of aroma precursors and damage the quality of tobacco leaves. This is similar to the photosynthetic characteristics of tobacco changed in response to cold stress.
Flue-curing mainly aimed to coordinate the water loss with yellowing of tobacco leaves. The tobacco leaves were yellowed in the ue-curing process because the rate of degradation of xanthophyll (such as carotenoid) was much lower than that of chlorophyll, therefore, tobacco leaves would turn from green to yellow. This indicated that the rates of water loss from tobacco leaves inside and outside the greenhouse in the ue-curing process varied slowly at rst, then rapidly changed and nally varied slowly again, which is consistent with existing research. The water loss rate reached its maximum at 42 to 48 °C. The water loss rate of tobacco leaves inside the greenhouse per unit time was lower than that outside. This may be due to the fact that the cell plasma membrane of tobacco leaves outside the shed was damaged after chilling stress, and they had wilting symptoms of varying degrees, so the water loss was faster. The tobacco leaves undergoing cold-damage presented a decreasing chlorophyll content; moreover, the thickened leaves, coverage of the wax coat, and the shrunk palisade tissues and spongy tissues caused tobacco leaves to be faced with di culties in water removal during ue-curing. As a result, the yellowing rate of tobacco leaves was greater than their rate of water loss, which caused the moisture content of laminae of tobacco leaves in the leaf-drying stage to remain high. With the further increase in temperature, cells of tobacco leaves were fractured and the cytosol owed out: on the one hand, the occurrence of the enzymatic browning reaction was accelerated; on the other hand, polyphenols were oxidized into black quinones on contact with air, which jointly led to generation of large-scale scalding of the tobacco.
The Effects of the Field Cold Stress on the Physiological and Biochemical Characteristics of Tobacco Leaves in the Harvest Period The change of the quality and curing characteristics of fresh tobacco leaves under cold stress was attributed to the fact that many free radicals damaged the plasma membrane system and disordered the activity of enzymes in tobacco leaves [28]. The results showed that the activities of POD, SOD and CAT of tobacco leaves in greenhouse were higher than those outside greenhouse, while the content and growth rate of MDA in tobacco leaves were lower than those outside greenhouse, which indicated that tobacco leaves outside greenhouse suffered obvious chilling stress, which led to the increase of cell membrane permeability and the destruction of physiological and biochemical environment. The curing process is a man-made stress environment. Under the same curing time, SOD, POD and CAT enzymes treated in the greenhouse can maintain high activity more effectively than those treated outside the greenhouse. As the main enzymes against reactive oxygen free radicals, they play an important role in alleviating cell senescence, which can re ect that the tobacco leaves in the greenhouse have stronger stress resistance than the tobacco leaves outside the greenhouse. Due to the damage of plasma membrane system after stress chilling injury of ue-cured tobacco, it is easy to cause the rapid combination of polyphenols in vacuoles and PPO in plastids in the curing process, thus reducing the quality of ue-cured tobacco leaves. The peak values of tobacco leaves inside and outside the greenhouse appeared at 42 °C and 48 °C respectively, indicating that the PPO of tobacco leaves outside the greenhouse was very active. It was possibly one of the more important reasons why scalded tobacco was more likely to be found after treatment outside the greenhouse subjected to cold stress than tobacco leaves inside the greenhouse.

The Effects of the Field Cold Stress on the Yield and Quality of Tobacco Leaves in the Harvest Period
Conventional chemical compositions, polyphenols and neutral aroma constituents in tobacco leaves are important factors determining the yield and the quality of the initially ue-cured tobacco leaves [29][30]. Cold stress affected the formation of the high-quality tobacco leaves during ue-curing. The result indicated that the content of carbon metabolites of tobacco leaves outside the greenhouse was signi cantly higher than that of tobacco leaves inside the greenhouse, while the content of nitrogen metabolites was on the contrary, indicating that the carbon metabolism of tobacco leaves outside the greenhouse was more active and nitrogen metabolism was inhibited within cold stress. Under cold stress, the photosynthetic system of the tobacco leaves was damaged to decrease root activity and physiological and biochemical enzyme activities [15]. The activation of carbon metabolism pathway is more bene cial to resist stress and alleviate. Under normal circumstances, in the later growth stage of ue-cured tobacco, nitrogen metabolism is inhibited, and carbon metabolism is damage to the plasma membrane system of ue-cured tobacco, which made it di cult to coordinate the water loss and yellowing of tobacco leaves during the curing process. These ndings will contribute to deal with the eld cold stress caused by high altitude conditions and to understand the browning tobacco leaves caused by various factors.

Experimental Materials
The experiment was carried out in Jianji village (E 99°33', N 26°31', at an altitude of 2565 m) in Laojun Mountain Town in 2019. The tobacco varieties HD were provided by Yuxi Zhongyan Tobacco Seed Co., Ltd., China. The seed is a conventional commercial tobacco seed. The tobacco variety HD was used for the experiment and was bred by utilizing oat breeding technology. The young seedlings bred under membranes were transplanted on 13 April, with the row and plant spacing being 120 cm × 60 cm. The seedlings were topped on 10 June, during which 15 or 16 pieces of the leaves were left; the lower leaves were picked and ue-cured on 3 July and ue-curing was completed on 7 September. The loam was used for the test, with the pH of 6.47, organic matter content of 56. 19

Experimental Design
Two treatments were set after tobacco leaves were topped. Treatment 1 (inside the greenhouse) was employed inside the plastic greenhouse: a spot with the length of 10 m and width of 5 m was randomly selected to establish a greenhouse made of steel frame, the top of which were covered with polyvinyl chloride (PVC) and polyethylene plastic for heat preservation, waterproof and light transmission. Two to three layers of shading nets distributed in the periphery of the greenhouse to preserve heat and keep out the wind, where the guard rows were set. Treatment 2 (outside the greenhouse) was conducted in the eld environment under natural conditions. A block with the same area as treatment 1 was stochastically selected, in the periphery of which guard rows were distributed. It needed the ensure that the two treatments were located in the same eld block. It also required to be guaranteed that at least 60 tobacco plants were grown in each treatment for normal repetition of the experiment. Moreover, a TH12R-EX recorder for humidity and temperature (Shenzhen Huahanwei Science and Technology Co., Ltd) was separately distributed inside and outside the greenhouse to record the changes of daily temperature and humidity. In addition, a WH-2310 wireless weather station (Jiaxing Misu Electronic Co., Ltd) was set outside the greenhouse to record a series of information including daily temperature, precipitation, and wind speed. Additionally, it was necessary to water the tobacco plants inside the greenhouse according to the natural precipitation outside the greenhouse.
The tobacco leaves were picked and waved in the conventional harvest period of middle leaves in the local area, to ensure the equilibrium and consistency of the maturity of tobacco leaves, and tobacco leaves in the same row should have same quality with moderate density. The tobacco leaves were ue-cured in a local bulk curing barn. The ue-curing was performed mainly according to the most advocated curing mode in the region (Fig. 10). Additionally, 100 to 120 tobacco leaves were weaved in each rod and totally three layers were set, in each of which there were 150 to170 rods.

Meteorological Data and Sample Collection
Through the weather station, the recorder for temperature and humidity, and the local meteorological bureau, the daily meteorological data (including temperature, rainfall and wind speed) in July and August were comprehensively collected. The meteorological data collected during the period when local tobacco leaves suffered eld cold stress were intensively analyzed. Only middle leaves (5th to 10th pieces of leaves from bottom to the top) of all tobacco Figure 2 Dynamic symptoms of tobacco after cold stress      Tobacco leaves of outside the greenhouse after curing.