Exogenous Foliar Melatonin Improves Cotton (Gossypium Hirsutum L.) Physio-Biochemical Characteristics Under the Synergetic Effects of Low Temperature and Salinity Stress

Low temperature and soil salinization during cotton sowing and seedling have adverse effects on cotton productivity. Finding an alternative for reducing the low temperature and salt induced damages during the seedling stage of cotton is a challenge for agricultural researchers nowadays. The physiological mechanism of exogenously applied melatonin (MT) on cotton seedlings under low temperature and salt stress is still unclear. The experiment in a phytotron was comprised with two temperature levels of 15°C and 25°C, and 5 MT treatments of 0, 50, 100, 150, 200 µM, and two salinity levels of 0 and 150 mM NaCl stress. Compared with the control treatments (non-salinity stress under 15°C and 25°C), the coupled stress of salt and low temperature reduced cotton seedlings’ biomass and net photosynthetic rate (P n ), aggravated the membrane damage, reduced the potassium (K + ) content and increased the sodium (Na + ) accumulation in the leaves and roots. Compared with the NaCl-stressed treatment alone, the exogenous foliar applications of 50-150µM MT signicantly increased the biomass and gas exchange parameters of cotton seedlings under the coupled salt and low temperature stress conditions. The exogenously applied MT at 50-150µM under the coupled effect of salt and low temperature stress conditions decreased the degree of membrane damage and regulated the activities of the protective enzymes, ion homeostasis, ion transport and absorption of cotton seedlings. The pairwise correlation analysis of each parameter by MT shows that the parameters with higher correlation with MT at 15°C are mainly malondialdehyde (MDA), peroxidase (POD), and catalase (CAT). The most relevant parameters at 25 ℃ are K + concentration in leaves (K + -L), K + concentration in root (K + -R), Na + concentration in leaves (Na + -L), Na + concentration in root (Na + -R), Na + uptake in-root surface (Na + -uptake), K + ion translocation (K + -translocation). Stepwise linear regression of the above parameters found that MT is more related to MDA at 15°C, and MT is more related to Na + -L at 25°C.


Introduction
Plants are often exposed to various environmental stresses, including drought, heavy-metal contamination, heat stress, low temperature, and soil salinization. Among these stresses, salinity and low temperature are important abiotic factors that are consequently associated with limited plant growth and productivity (Ashraf and Foolad, 2007;Mishra et al., 2016;Sun et al., 2017;Naing and Kim, 2021). Moreover, over 50% of the world's arable land is predicted to be affected by salinity by 2050 (Demiral and Türkan, 2006;Saeed et al., 2021), while 15% of the suitable agricultural areas are affected by temperature stress worldwide (Çiçek and Çakırlar, 2008). Soil salinization is often accompanied by temperature stress because changes in ambient temperature are more frequent than changes in other abiotic factors. On the other hand, changes in ambient temperature rapidly aggravates other environmental stresses, including salinity (Ashraf and Foolad, 2007;Çiçek and Çakırlar, 2008).
Salinity caused by NaCl has recently become a focus in environmental research investigations. NaCl-associated salinity leads to a wide range of changes in plant metabolism (Dong et al., 2014;Hamani et al., 2021). An increasing NaCl concentration affects plants in several ways. It causes nutrient de ciencies, osmotic stress and speci c ion toxicity, thereby affecting several physiological mechanisms involved in plant metabolism (Munns, 2002). Salt stress affects a range of important mechanisms, including photosynthesis, energy and lipid metabolism and protein synthesis (Parida and Das, 2005). Low temperature stress generally affects plant growth and induces reactive oxygen species production, leading to damage of cell membrane structure (Yan et al., 2021). Previous studies reported that low temperature stress rapidly increases antioxidants activities, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Airaki et al., 2012;Sun et al., 2017). Lichtenthaler et al. (2005) founded that the photosynthetic functions and physicochemical properties of thylakoid membranes are directly or indirectly affected by temperature and salt stresses.
Melatonin (N-acetyl-5-methoxytryptamine), which was initially identi ed and isolated from the pineal glands of cows, is an important multifunctional hormone that involved in a range of animal's physiological processes modulation, such as sleep (Zhao et al., 2022), immunity and reproduction (Arnao and Hernández-Ruiz, 2006;Calvo et al., 2013), circadian rhythm and antioxidant activity (Zhan et al., 2021). Melatonin was discovered for the rst time in 1995 in vascular plants as an indoleamine hormone (Hattori et al., 1995). Melatonin plays a key role plant growth and development by promoting root growth and seedling's biomass accumulation, like auxin in function (Byeon and Back, 2014;Hardeland, 2016). Several researches revealed the importance of melatonin in mitigating biotic and abiotic stresses (Liang et al., 2015;Shi et al., 2015;Sharma et al., 2020). Known as an antioxidant and free radical scavenger, exogenously applied melatonin improved plants' resistance to biotic and abiotic stresses, which confers to plant stress resistance by enhancing photosynthesis, ion homeostasis and antioxidant enzymes activities (Bajwa et al., 2014;Zhang and Zhang, 2014). Exogenous foliar application of melatonin is involved in numerous physiological processes to improve plant resistance to salinity stress (Zhao et al., 2022). Recent study demonstrated that exogenously applied melatonin could sustain a high photosynthetic rate in tea plants, which enhances salt tolerance through its effects on antioxidant response against environmental stress (Li et al., 2019). Exogenous application of melatonin under sat stress showed alleviating effects on horticultural crops, such as increase in primary root length and antioxidant activity in sun ower (Mukherjee et al., 2014), increase in net photosynthetic rate and stomatal conductance in tomato (Ding et al., 2017), and improving photosynthetic e ciency, endogenous melatonin content and ion homeostasis in upland cotton . On the other hand, exogenous application of melatonin was found to improve the resistance capacity of horticultural crops to low temperature stress by, increasing photosynthetic rate, antioxidant activity, and decreasing MDA content in tea plants , increasing endogenous melatonin content, antioxidant activity and decreasing malondialdehyde (MDA) content in Bermudagrass (Fan et al., 2015), and increasing photosynthetic carbon assimilation, activities of antioxidant enzymes, levels of non-enzymatic antioxidants and decreasing in cold damage on cell membranes accumulation of ROS in tomato (Ding et al., 2017).
Cotton is the world's most important natural textile ber with an incredible economical value and is considered as the backbone of the textile industry. Despite the capacity of cotton plants to relatively tolerate environmental stress, exposure to high stress condition could negatively affect cotton growth, development, and ultimately yield Hamani et al., 2020). For cotton grown in salty soils in arid/semiarid region, cotton seedlings often suffer environmental stress of low temperature and salinity. Therefore, several investigations have aroused keen interest for improving cotton resilience. Previous studies revealed mechanism of exogenous melatonin on crop resilience improvement under alone stress of salinity and low temperature, while there was little information on the effects of exogenous melatonin on crop performance under coupled low temperature and NaCl-stressed. Therefore, the main purpose of this study was to determine the physiological and biochemical responses of low temperature and NaCl-stressed cotton seedlings sprayed with exogenous melatonin.

Plant materials
The cotton variety, Xinluzhong37, was purchased from Tahe Seed Industry Co., Ltd., Alar City. It is a mid-early maturing upland cotton variety with a growth period of about 140 days. Melatonin (MT), sodium chloride (NaCl), sodium sulfate (Na 2 SO 4 ) and other chemical reagents are all analytically pure, purchased from Beijing Soleibao and Sinopharm Chemical Reagent Co., Ltd.

Experimental design
The experiment was carried out in a phytotron in the Xinxiang Comprehensive Experimental Station of Chinese Academy of Agricultural Sciences (35.09°N, 113.48°E and altitude 81 m). The humidity of the climate room is 40-50%, the photoperiod is 12 h, the light intensity is 600 µmol m −2 s −1 (lighting is provided by LED lights), and the temperature is controlled according to the experimental design. The experimental design is shown in Table 1.  Uniform size and plump cotton seeds were selected, disinfected with 1000 times' diluted solution of carbendazim, and rinsed with deionized water after 30 minutes. The sterilized cotton seeds were sown at a depth of 2 cm in PVC pots (diameter 6 cm, height 24 cm) containing 780 g of ne sand (sand was sterilized by high temperature). After sowing, the PVC pots were cover with an opaque shading board to keep the surface of the sand moist and facilitate the germination of seeds. For germination, the PVC pots were placed in a phytotron with a day/night temperature of 25/20°C. After the seeds germinated, the shading was removed from the pots. When the seedlings grow to 1 true leaf, days with about 80 ml of the 1/2 Hoagland solution. When the seedlings grown to two leaves and one heart leaf, they were randomly divided into groups for starting the salt and temperature treatments. The cotton seedlings treated at room temperature (25˚C) and low temperature (15˚C) were placed in phytotrons with day/night temperatures of 25/18°C and 15/8°C, respectively. On the 1st, 4th, and 9th day under the temperature treatment, seedlings were irrigated with 150 mmol L −1 salt water (NaCl: Na 2 SO 4 =9:1) in the morning. Every day after turning off the lights, different concentrations of MT (0, 50, 100, 150, 200 µmol L −1 ) were sprayed on the upper surface of the leaves of salt and low temperature-stressed cotton seedlings. On the 10th day, the fully expanded leaves were sampled for the measurement of various indicators.

Determination of gas exchange parameters
The LI-6400XT photosynthesis measuring system (Licor, USA) was used to measure, the net photosynthetic rate (P n ), stomatal conductance (g s ) intercellular carbon dioxide concentration (C i ), and transpiration rate (T r ) of the fully expanded leaves of cotton seedlings between 9:00-11:00 in the morning. For the gas exchange parameters measurements, 3 plants were randomly selected from each treatment, the red and blue light source leaf chamber was used, and the light intensity was maintained at 1000 µmol·m −2 ·s −1 , and the CO 2 concentration in the sample chamber was 400 µmol·mol −1 (using Li-6400-01 Liquefaction small steel bottle to control the CO 2 concentration in the sample chamber), block temperature is 25°C, and air ow is 500 µmol s −1 . During the measurement period, tubes containing desiccant and soda were used to control H 2 O and CO 2 , respectively inside the instrument chamber.

Determination of cotton seedling biomass
Three plants were randomly selected for each treatment, the shoots were separated from the roots, and the fresh weights were respectively weighed and put into sample bags. The samples were put into a sample bag at 105°C for 30 min and oven-dried at 75°C for 48 h, the dry weight was then measured.
2.3.3. Determination of MDA and superoxide anion About 0.3 g of fresh leaf tissue was ground using a pestle and mortar. An amount of 3.0 mL of 0.05 M precooled phosphate buffer (pH 7.8) was added to the homogenate, which was then centrifuged at 15000 g at 4°C for 20 min. Lipid peroxidation was measured spectrophotometrically from MDA content using a thiobarbituric acid (TBA) reaction following the method described by Heath and Packer (1968). At the same time, the same supernatant Hydroxylamine was used to determine the superoxide anion production rate of leaves (Tian et al., 2003).

Determination of protective enzyme activity
About 0.3g of fresh leaf tissue was ground using a pestle and mortar. An amount of 2.7 mL of 0.05 M precooled phosphate buffer (pH 7.8) was added to the homogenate, which was then centrifuged at 15000 g at 4°C for 20 min. After obtaining the supernatant, refer to the following method to determine the protective enzyme activity. SOD activity was determined spectrophotometrically from the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm (Dhindsa et al., 1981). POD activity was measured by the determination of guaiacol oxidation by H 2 O 2 at 470 nm (Lacan and Baccou, 1998

Determination of ion content
On the 10th day after the treatment, 3 cotton seedling leaves and roots were randomly selected for each treatment, oven-dried at 105°C for 30 min, and then continued drying at 75°C for 48 h. The dried leaves and roots were ground into powder. Each sample weighed at 0.1500 g, digested with H 2 SO 4 -H 2 O 2 , and diluted in a 100 ml volumetric ask. The ltered supernatant was used to determine the sodium (Na + ) and potassium (K + ) ion contents in the leaves and roots of cotton seedlings using a ame photometer. Na + uptake at the seedlings' root surface and K + translocation from root to shoot were computed by following the equations described by (Malik et al., 2010);Shabala and Mackay (2011), respectively.

Statistical analysis
The test data is statistically analyzed using EXCEL 2020 and DPS V13.5 softwares. All experimental data were expressed as means ± standard deviation. All treatment means (n = 3) were compared for any signi cant differences using Duncan's multiple range tests at P< 0.05. Data tting and graphical presentation were carried out in Origin-Pro 2021a (Origin Lab, Northampton, MA, USA). A general linear regression model was used to t the relationships between parameters.

The effect of MT on cotton seedling biomass under low temperature and salt stress
Compared with CK, the coupled stress of salt and low temperature reduced the dry weights of shoot and root of cotton seedlings (Figure 1ab), but the reduction of root dry weight did not reach a signi cant level. Under the salt stress condition at 25℃, although the aboveground dry weight of cotton seedlings treated with exogenous MT increased slightly (compared with 0 µM MT), it was not signi cant; and there was no signi cant difference between different MT treatments. Except for 200 µM MT, exogenous MT signi cantly increased the root dry weight of cotton seedlings, and the highest value of cotton root dry weight was obtained in the 150 µM MT treatment. Under the coupled stress of low temperature and salinity, exogenously applied MT insigni cantly increased the aboveground and root dry weights of cotton seedlings; the maximum aboveground dry weight was obtained in the 100 mM MT treatment.
3.2. Effects of MT on gas exchange parameters of cotton seedling leaves under low temperature and salt stress Compared with CK, the coupled stress of low temperature and salinity signi cantly reduced P n , g s , T r , and C i of cotton seedlings ( Figure 2).
Under the salt stress condition at 25℃, with the increase of exogenous MT concentration, P n , g s , T r , and C i all showed a trend of increase rstly and then decreased in the 200µM MT treatment compared with the non-MT treated saline treatment. Except for C i , the highest values of gas exchange parameters appeared in the 150µM MT treatment. Under the coupled stress of low temperature and salinity, the application of different concentrations of exogenous MT increased P n , g s , and T r of cotton seedlings, and with the increase of MT concentration, P n , g s , and T r also showed a trend of increasing and then decreasing, but the difference between MT concentrations is not signi cant. The highest value of P n was obtained with the 100 µM MT treatment.
3.3. The effect of MT on cotton seedling membrane damage under low temperature and salt stress Compared with CK (0 mM NaCl), low temperature + salt stress insigni cantly increased the superoxide anion production rate and MDA content in cotton seedling leaves (Figure 3). At 25℃, the exogenously applied MT reduced the superoxide anion production rate and MDA content in cotton seedling leaves under salt stress. Compared with the MT-untreated saline treatment, the superoxide anion production rate was reduced by 29.6% under the 100 µM MT treatment, and the MDA content was signi cantly reduced by 43.  Table 1 shows the effect of exogenously applied MT on the antioxidant enzymes of cotton seedlings under low temperature and salt stress.
Compared with CK (0 mM NaCl), salt stress signi cantly reduced the SOD and POD contents in cotton seedling leaves (38% and 31%), while increased the CAT content (100%). The coupled stress of low temperature and salt signi cantly reduced the POD and CAT contents (67% and 96%), slightly increased the SOD content. Compared with the MT-untreated saline treatment at 25℃, exogenously sprayed MT signi cantly reduced the SOD and POD contents and insigni cantly affected the CAT concentration in seedlings leaves. Compared with the MT-untreated saline treatment under the coupled stress of low temperature and salt, spraying 50-100 µM MT on the leaves of seedlings increased the SOD and POD contents, and decreased the CAT content. Compared with 0 µM MT salt treatment under the dual stress of low temperature and salt, spraying 50-100 µM MT on the leaves increased the SOD and POD contents, and decreased the CAT content.
Through an interactive analysis between the treatments, it was found that different temperatures conditions had a signi cant effect on the SOD and CAT contents, but had an insigni cant effect on POD of cotton seedling leaves under salt stress. Different MT concentrations had a signi cant impact on POD and CAT, while has an insigni cant effect on SOD of cotton seedlings under salt stress. The content of POD and CAT in the leaves of cotton seedlings was affected by the interaction of temperature and MT.
3.5. The effect of MT on ion homeostasis and absorption under different temperature and salt stress Figure 4 shows the effect of low temperature + salt stress on the ion homeostasis of cotton seedlings. The results showed that compared with the control, salt stress reduced the K + content in the seedlings leaves and roots by 15% and 50%, and increased the Na + content by 422% and 249%, respectively. The coupled stress of low temperature and salt slightly increased the K + content in the leaves, signi cantly reduced the K + content in the roots, and signi cantly increased the accumulation of Na + in the leaves and roots. After foliar spraying of different concentrations of MT, the K + content in the roots under salt stress conditions was signi cantly increased, and the K + content in the leaves of cotton seedlings increased slightly. The Na + content in the leaves and roots of cotton seedlings was signi cantly reduced, but the difference between different concentrations of MT was insigni cant. Compared with the MT-untreated saline treatment, the application of 50 µM MT under salt stress, slightly increased the K + content in the leaves and roots of cotton seedlings, and different concentrations of MT signi cantly reduced the content of cotton seedlings. The content of Na + , although the amount of Na + in the root decreased, the difference was insigni cant.
Compared with the control treatment, the coupled stress of low temperature and salt, signi cantly increased the Na + /K + ratio in the leaves and roots of cotton seedlings (542% and 289%, respectively) (Figure 5a,b), and signi cantly increased the Na + content on the surface of cotton seedling roots (585%) (Figure 5d). Exogenous foliar spraying of different MT concentrations signi cantly reduced the Na + /K + ratio in the leaves and roots of cotton seedlings under salt stress at 25℃, and correspondingly reduced the Na + absorption on the surface of cotton seedling roots. Under the coupled stress of low temperature and salt, with the increase of MT concentration, the Na + /K + ratio in the leaves of cotton seedlings rstly decreased and then increased. The Na + /K + ratio in the roots increased and then decreased, correspondingly reducing the root surface absorption of Na + . On the other hand, compared with the control, the coupled stress of low temperature and salt signi cantly increased the transfer of K + ion from roots to shoot and then from shoot to leaves of cotton seedlings. After exogenously foliar spraying of different concentrations of MT, the low temperature + salt stress signi cantly increased K + transport from roots to aboveground parts.
Compared with the MT-untreated saline treatment, different concentrations of MT slightly reduced the transport of K + from roots to shoot.

Discussion
Abiotic stresses such as salinity and low temperature severely limit the growth and development of plants. Moreover, nearly 50% of the annual yield loss of major crops worldwide is related to abiotic stresses (Valliyodan and Nguyen, 2006 2016b). In this study, the combination of low temperature + salinity stress has a greater impact on cotton seedling biomass than the single salt stress or single Low temperature stress. This is mainly related to the senescence and dehydration of plant cells under abiotic stress, and salinity limits cell elongation and division, which leads to a decrease in the growth rate of roots and aboveground parts, and a decrease in dry matter accumulation (Khalil et al., 2022).
Photosynthesis is the basic source of the existence, prosperity and development of life on earth. Photosynthesis is the basis of plant growth and development, but it is susceptible to environmental stresses (particularly sensitive to low temperature stress). The main components of photosynthesis (electron transport, Calvin cycle, stomatal conductance) were destroyed under low temperature stress (Hu et al., 2016). In this study, g s and T r decreased in response to salinity alone, low temperature alone and salinity + low temperature and plants reduced water loss by closing their stomata. The closing of stomata leads to insu cient CO 2 and inhibits photosynthesis. This is consistent with the research results of Chatrath et al. (2000), who reported that plant may close their stomata under saline conditions in order to reduce water loss. After exogenous foliar supplementation with MT, the aboveground and underground biomass under the different stresses of salinity, low temperature, and salinity + low temperature was increased. This is related to the ability of MT to regulate the physical process of cell wall extension to induce plant root growth and promote the accumulation of dry matter (Sarropoulou et al., 2012). After the foliar application of MT, the g s and T r of cotton seedlings were increased, thereby increasing their P n . This is consistent with the research results of Irshad et al.
(2021) and Zhang et al. (2021), who observed that exogenous MT has signi cantly improved P n under low temperature stress and salt stress, respectively.
The normal transportation of substances and ions in plants mainly depends on the integrity of the structure and function of the bio lm. When the plant is subjected to abiotic stress, plant's bio lm system is the rst to be impacted and damaged. One of the damages caused by abiotic stress to cell membranes is lipid peroxidation. The nal product of peroxidation damage to cell membranes by reactive oxygen species is MDA. The production of reactive oxygen species (ROS) such as H 2 O 2 , superoxide anion, hydroxyl anion, and MDA under salt conditions severely destroys chlorophyll content, destroys lipids and mitochondria, and causes plant necrosis (Mohsin et al., 2020). In this study, under the different stresses of 150 mM NaCl, 15℃ low temperature and 150 mM NaCl + low temperature, cotton seedlings MDA content increased compared with the control, and at the same time the superoxide anion production rate increased, which led to membrane lipid peroxidation. The research results are consistent with those of Parveen et al. (2021). Abiotic stress causes an increase in the level of active oxygen in the plant body and simultaneously activates the defense system to reduce the harm of active oxygen. SOD, CAT and POD are the key enzymes to remove active oxygen in cells, and the ability to remove active oxygen depends on the coordination of these enzymes (Hernandez et al., 2000). As the rst line of defense for ROS removal, SOD can disproportionate superoxide anions into O 2 and H 2 O 2 , and POD and CAT can remove H 2 O 2 in peroxisomes (Anjum et al., 2017). In this experiment, compared with the control, the coupled stress of salt and low temperature and salt stress increased the content of CAT and decreased the content of SOD and POD, while the low temperature stress increased the content of POD and CAT, and decreased the content of SOD. MT can increase the activity of protective enzymes to alleviate the damage caused by abiotic stress to plants. Studies have shown that the application of exogenous MT can increase the content of SOD, POD and CAT in cucumber under salt stress environment (Wang et al., 2016), increase the content of SOD in wheat (Turk et al., 2014) and cucumber (Marta et al., 2016) under low temperature stress. In addition, under salt and heat stress, applying MT increased tomato CAT content and reduced SOD content (Martinez et al., 2018). In this experiment, after applying the exogenous MT, compared with the salt treatment without the exogenous MT, the exogenous spraying of MT at a concentration of 50 µM increased the POD and CAT content of cotton seedlings, while at low temperature + Under salt double stress, spraying 50-150 µM MT signi cantly increased the SOD and POD content of cotton seedlings. Through interactive analysis, it is known that temperature has a very signi cant impact on SOD and CAT, and MT has a very signi cant impact on POD and CAT. At the same time, POD and CAT are affected by the interaction of temperature and MT.
Under salt stress, the excessive accumulation of Na + in plant cells leads to the production of ROS, which breaks the dynamic balance of the scavenging system, and initiates membrane lipid peroxidation and degreasing. The membrane structure is destroyed and the MDA content increases (Hernandez et al., 2000). In this study, after exogenous foliar supplementation with MT under salt stress, the opposite conclusion was reached and found that the Na + content in cotton seedlings was reduced, the protective enzyme activity was increased, the degree of membrane damage was reduced, and the photosynthetic parameters and dry matter content were increased. The reason is that exogenous MT regulates the physiological mechanism of cotton seedlings to resist adversity under salt stress. As shown in Figure 5a, except that the protective enzyme activity of cotton seedlings unilaterally affects photosynthetic parameters. Biomass is not directly affected by photosynthetic parameters and membrane damage and other parameters have no mutual in uence. Previous study reported that the high accumulation of Na + in the apoplasts of roots and stems easily triggers osmotic stress (Zhao et al., 2020). The higher accumulation of Na + leads to an increase in the ratio of Na + /K + ratio and promotes ion imbalance, thereby hindering metabolic functions and destroying plant activities (Mohsin et al., 2020). For example, photosynthesis declines and dry matter accumulation decreases, which ultimately leads to reduced crop growth (Sarropoulou et al., 2012), production of reactive oxygen species , and plant damage. The results of this study showed that, compared with the control, the different stresses of salinity and low temperature + salinity reduced the K + accumulation in the leaves and roots of cotton seedlings, and signi cantly increased the accumulation of Na + , resulting in a signi cant increase in the ratio of Na + /K + , and at the same time increased the absorption of Na + . However, after spraying the exogenous MT, the Na + accumulation in the leaves and roots of seedlings was signi cantly reduced. The K + accumulation in the leaves of the seedlings was increased, and the Na + /K + ratio of seedlings was reduced. Since the K + dynamic balance is affected by the accumulation of Na + , an appropriate Na + /K + should be maintained to improve the salt tolerance of crops (Rasouli et al., 2021). Studies have found that under abiotic stress, plants closing stomata and reducing transpiration may limit the transport of Na + to plant tissues (Perin et al., 2019). In the current study, through interactive analysis, temperature signi cantly affects K + , Na + , Na + /K + ratio, Na + absorption and K + transport in roots and leaves, except for the insigni cant effect of temperature on the Na + content in leaves. The in uence of exogenous MT on K + content is insigni cant. The Na + content in root removal is not affected by the interaction of temperature and MT, and the others are all affected by the interaction of temperature and MT.
Studies have shown that low temperature stress can enhance the antioxidant defense ability by regulating the metabolic and photosynthetic reactions of tea plants, wheat, Arabidopsis and other plants, thereby improving the cold tolerance of plants (Yu et al., 2018;Li et al., 2019). The results of this study are that when exogenous MT is applied under low temperature + salt stress, the ion content directly affects the protective enzymes, photosynthetic parameters, membrane damage and biomass, while the protective enzymes and photosynthetic parameters negatively affect the ion content. Protective enzymes affect membrane damage in one direction, and have a two-way effect on photosynthesis ( Figure 6). In summary, through interactive analysis, temperature has a signi cant effect on the aboveground and underground biomasses under salt stress conditions. Exogenous MT successfully increased the biomass and physiological and biochemical indexes of cotton seedlings under the stresses of salt and low temperature + salt. In addition, reducing the absorption of ROS and Na + in plants is the focus of this study, indicating that exogenous MT has the potential to store crop growth under salt stress. The relationships between various parameters of cotton seedlings at 15°C and 25°C under salt stress condition were shown in Figure 7 and Table 3. It was observed that improving antioxidant enzymes activities with exogenous MT turns to an improvement of leaf gas exchange parameters and K + uptake, while decreased Na + uptake. concentration; Tr, transpiration rate; K-L, leaf K + content; K-R, root K + content; Na-L, leaf Na + content; Na-R, root Na + content; Na + uptake; K + translocation.
In summary, exogenous melatonin has different regulatory mechanisms on the ion content, photosynthetic parameters, membrane damage, protective enzymes activities and biomass accumulation of cotton seedlings under coupled salt and low temperature stress ( Figure 6). Protecting enzymes under salt stress affects photosynthesis in one direction, while under low temperature and salt stress the two interact with each other. The protection enzyme activity under salt stress affects membrane damage one-way, ion content one-way affects membrane damage and biomass, while under low temperature and salt dual stress, the protection enzyme and membrane damage, ion content and membrane damage and biomass are two-way effects. Under double coercion, MT's adjustment of the above parameters is more complex.

Conclusion
The results showed that under salt stress and coupled low temperature and salt stress, the spraying of 50 to 150 MT on the leaf surface increased the biomass, P n , and SOD and POD activities, while reduced membrane damage of cotton seedling leaves. As a result, the absorption of K + reduces the accumulation of Na + . The regulatory mechanism of external supplementary MT on salt stress and salt stress and low temperature stress is different. The regulation of the mechanism under low temperature and salt stress is more complex. In this study, the lack of MT alleviates the abiotic stress mechanism of cotton seedlings in a more in-depth study, the next step can focus on the study of the metabolic pathway changes of cotton seedlings under abiotic stress by the exogenous substance MT.   Effects of exogenous Melatonin (MT) on (a) Superoxide anion and (b) malondialdehyde (MDA) of cotton seedlings under 150 mM NaClstressed and low-temperature condition. Values are means ± standard deviation (n = 3). Different letters represent signi cant differences at P < 0.05.

Figure 4
Effects of exogenous Melatonin (MT) on (a) K+ concentration in the leaves, (b) K+ concentration in the roots, (c) Na+ concentration in the leaves, and (d) Na+ concentration in the roots of cotton seedlings under the 150 mM NaCl-stressed and low-temperature conditions. Values are means ± standard deviation (n = 3). Different letters represent signi cant differences at P < 0.05.

Figure 5
Effects of exogenous Melatonin (MT) on (a) Na+/K+ ratio in plant leaves, (b) Na+/K+ ratio in plant roots, (c) K+ ion translocation (root to shoot), and (d) Na+ uptake in-root surface of cotton seedlings under the 150 mM NaCl-stressed and low-temperature condition. Values are means ± standard deviation (n = 3). Different letters represent signi cant differences at P < 0.05.

Figure 6
Effects of exogenous MT on the physiological mechanism of cotton seedlings under (a) salt stress and (b) under coupled low temperature and salt stress. Yellow double orientation arrow indicates that the two affect each other; the red one-way arrow indicates unilateral in uence pointing.