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Foliar application of 24-epibrassinolide enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco by regulating cold stress tolerance

BMC Plant Biology volume 25, Article number: 77 (2025) Cite this article

Abstract

Background

Low temperatures disrupt nitrogen metabolism in tobacco, resulting in lower nicotine content in the leaves. 24-epibrassinolide (EBR) is a widely used plant growth regulator known for its roles in enhancing cold tolerance and nitrogen metabolism. Nevertheless, it remains unclear whether EBR enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco.

Results

To investigate the effects of EBR on leaf nicotine content under low temperature conditions during the mature stage of ‘Yunyan 87’ flue-cured tobacco, four treatments (foliar spraying of 0, 0.1, 0.2 and 0.4 mg·L− 1 EBR solutions) were performed by using a single-factor randomized complete block design. The result showed that foliar spraying of different concentrations of EBR notably improve the agronomic and economic traits of flue-cured tobacco to varying degrees, as well as increase the total nitrogen and nicotine content in the tobacco leaves. 0.2 mg·L− 1 EBR treatment showed better results, with nicotine content in the middle and upper leaves after curing increasing by 11.11% and 19.90%, respectively, compared to CK. Compared to the single EBR, foliar spraying of EBR compound containing α-Cyclodextrin and Tween 80 prolongs the effect of EBR, promotes the growth and development of tobacco plants. Combining EBR with CaCl2 and ZnSO4·7H2O significantly enhances the cold resistance of tobacco plants. Furthermore, combining EBR with higher concentrations of KH2PO4 is more effective in promoting the maturation and yellowing of the upper leaves than those with lower concentrations.

Conclusions

This study provides new insights that foliar application of EBR enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco by regulating cold stress tolerance. The integration of EBR with α-Cyclodextrin, Tween 80, CaCl2, ZnSO4·7H2O and KH2PO4 showcases a novel approach to extending the effectiveness of plant growth regulators and improving agricultural sustainability. Furthermore, these findings may be applicable to other cold-sensitive crops, offering broader benefits for improving resilience and productivity under low temperatures. However, the research focuses on two growth cycles, without investigating the long-term impact of EBR on soil health, crop sustainability, and ecosystem. And further research is needed to elucidate the molecular mechanisms of EBR on enhancing leaf nicotine content.

Clinical trial number

Not applicable.

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Background

Nicotine, 1-methyl-2-(3-pyridyl)-pyrrolidine, is nitrogenous organic compounds produced in Nicotiana species [1]. As a secondary metabolite, nicotine is produced by tobacco plants in response to external stress [2]. Nicotine is primarily synthesized in the roots and transported to the leaves via the xylem, where it accumulates [3]. Natural nicotine and its derivatives are valuable for their applications in medicine, agriculture and chemical industry [4,5,6,7,8]. Most commercial nicotine is extracted from tobacco plants. However, extraction yields are often low and costs high due to factors like genetic factors [9,10,11], cultivation practices such as fertilization and topping [12,13,14,15,16,17,18], as well as environmental factors like ultraviolet exposure [19], carbon monoxide levels [20], and temperature [21]. There is a positive correlation between leaf nicotine content and local temperatures (14–30 °C) [21]. In high-altitude regions, flue-cured tobacco frequently encounters low temperatures and rainy weather during the late growth stages. These conditions delay leaf harvest and reduce the nicotine content of the leaves, which hinder large-scale industrial production. Consequently, research into optimizing nicotine regulation has become increasingly important.

Low and non-freezing temperatures affects the productivity, survival, and ecological distribution of crop species by injuring and killing crop species [22]. As sessile organisms, plants must endure unfavorable environmental conditions by developing effective survival strategies [23]. Through evolution, plants have developed the ability to withstand seasonal low temperatures (0–15℃) without freezing, a capability known as cold stress tolerance [24]. Tobacco, a thermophilic economic crop, is particularly vulnerable to low temperatures [25]. During seedling stage, tobacco leaves exhibit wilt and shrinkage when subjected to cold stress, leading to inhibited growth. This stunted growth includes reduced plant height and fewer effective leaves, and can lead to early flowering [26]. Moreover, cold stress alters the quality of tobacco leaves by affecting their microstructure, chlorophyll content, and maturity, thereby compromising the curing characteristics of leaves, ultimately reducing the quality of cured tobacco leaves [27,28,29]. To cope with cold stress, tobacco seedlings are cultivated in greenhouses, significantly enhancing cold tolerance during the seedling stage. Protective coverings further reduce the impact of low temperatures on plants. Additionally, proper management practices, including fertilization, irrigation, and drainage, enhance the cold tolerance in tobacco plants. However, these measures are costly to implement on a large scale. Therefore, it is worth exploring whether the use of cost-effective external substances can improve the cold tolerance of tobacco.

Brassinosteroids (BRs), the sixth class of plant hormones, play a crucial role in regulating plant growth and development. They also help plants respond to both biotic and abiotic stresses, enhance photosynthesis, and influence the yield of horticultural crops as well as the quality of produce during post-harvest storage [30]. To date, over 70 different BRs have been identified across various plant species. Among them, brassinolide (BL), 24-epibrassinolide (EBR), and 28-homobrassinolide (HBL) are noted for their high biological activity [31]. EBR is a widely used synthetic brassinosteroid known for regulating plant growth [32]. Spraying with exogenous EBR promotes photosynthesis and growth by enhancing activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and expression of photosynthetic genes in cucumber seedlings [33]. Application of EBR could also enhance the cold tolerance by reducing membrane injury index and increasing total chlorophyll, soluble sugar and protein content in maize seedlings [34]. Behnamnia’ study showed that spraying with exogenous EBR alleviates drought-induced oxidative stress by decreasing the MDA content and enhancing the activity of antioxidant enzymes in tomato seedlings [35]. EBR could also boost nitrogen metabolism among different plants [36,37,38]. For example, Spraying with EBR promotes nitrogen absorption and assimilation efficiency of apple seedlings under salt stress [36]. However, these studies primarily examine the impact of EBR on the seedling stage of plants, while it remains unexplored the effects of EBR on these aspects during the mature stage. In addition, limited research shows that exogenous EBR increases nicotine content in tobacco leaves. Therefore, it needs to explore whether EBR enhances the leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco.

Cyclodextrin and Tween are known as pesticide stabilizers, regulating the release rate of pesticides and enhance their stability [39, 40]. As antifreezes, calcium chloride (CaCl2) and zinc sulfate (ZnSO4) can mitigate plant damage and improving cold tolerance under low temperature conditions [41, 42]. Potassium dihydrogen phosphate (KH2PO4), a phosphorus-potassium complex, is commonly used in foliar fertilization to supply plants with phosphorus and potassium. Recent studies have demonstrated that foliar application of KH2PO4 notably enhances chlorophyll content and net photosynthetic rate in wheat flag leaves, resulting in increased thousand-grain weight and dry matter yield [43]. Additionally, KH2PO4 can improve chlorophyll content, osmoregulation, and antioxidant capacity in rice [44]. In tobacco, KH2PO4 was found to promote the yellowing and maturation of upper leaves [45,46,47]. As a major source of nicotine, flue-cured tobacco cultivar ‘Yunyan 87’ was used to investigate the effects of different concentrations of EBR, EBR compounds with pesticide stabilizers (α-Cyclodextrin and Tween 80), antifreezes (CaCl2 and ZnSO4·7H2O), and ripener (KH2PO4) on the agronomic and economic traits, chemical components content, and especially leaf nicotine content. This study aims to establish a theoretical framework for using exogenous EBR to enhance leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco by regulating cold stress tolerance.

Materials and methods

Experimental location and materials

The experiment was conducted from 2021 to 2022 in liangjiazhuang village, zhuyang town, Lingbao city, Sanmenxia city, Henan province. The site, at an altitude of 989 m, previously grew tobacco. The soil type is loam, with a pH of 7.55, containing 15.76 mg·kg− 1 organic matter, 82.56 mg·kg− 1 available nitrogen, 52.94 mg·kg− 1 available potassium, and 16.24 mg·kg− 1 available phosphorus.

The tobacco (Nicotiana tabacum L.) cultivar utilized in this experiment was ‘Yunyan 87’. ‘Yunyan 87’ is developed by the Yunnan Tobacco Agricultural Science Research Institute through hybridization of ‘Yunyan 2’ as the female parent and ‘K326’ as the male parent, followed by pedigree selection [48]. The seeds of ‘Yunyan 87’ were provided by Sanmenxia Branch of Henan Provincial Tobacco Corporation, China. Seedlings were produced using floated technology and transplanted on May 12. The EBR stock solution (0.1 g·L− 1) was provided by Hebei Lansheng Biotechnology Co., Ltd. α-cyclodextrin and Tween 80 were produced by Shanghai Macklin Biochemical Technology Co., Ltd. Calcium chloride (CaCl2) and Zinc sulfate heptahydrate (ZnSO4·7H2O) were produced by Sinopharm Chemical Reagent Co., Ltd. Potassium dihydrogen phosphate (KH2PO4) was produced by Sichuan Run er Technology Co., Ltd. The tested organic-inorganic bio-fertilizer (organic matter content (mass fraction) ≥ 40%, N: P = 10:12:18) was produced by Sanmenxia Longfei Bioengineering Co., Ltd. Fermented soybean powder was produced by Sanmenxia Xinding Agricultural Technology Co., Ltd. Potassium sulfate was produced by Guotou Xinjiang Lop Nur Potash Co., Ltd., potassium nitrate was sourced by Shanxi Jinlan Chemical Co., Ltd., and superphosphate was obtained from Luoyang Qi He Ecological Technology Co., Ltd.

Experimental treatments

Effects of applying different EBR concentrations on nicotine content in tobacco leaves at maturity in low-temperature regions

This experiment employed a single-factor randomized complete block design with four treatments: CK1 (250 mL water), A1 (0.1 mg·L− 1 EBR solution), A2 (0.2 mg·L− 1 EBR solution), and A3 (0.4 mg·L− 1 EBR solution) per plant. Conducted in 2021, the experimental plot covered 60 m², with each plot containing 4 rows and 100 tobacco plants, spaced 1.2 m × 0.5 m. The study included three field replicates, following local guidelines for high-quality flue-cured tobacco production. On July 21, plants with uniform growth were topped when the first central flower of tobacco plants reached 50% bloom, leaving 20 leaves per plant. Foliar spraying treatments were then applied using a manual sprayer to evenly coat both leaf surfaces. Agronomic traits were measured on three representative plants at 0, 15, and 30 d post-treatment. After curing, 1.00 kg of middle (C3F) and upper (B2F) leaves were collected from each treatment group for chemical composition analysis.

Regulatory effect and mechanism of EBR compounds on nicotine content in mature tobacco leaves

This experiment also employed a single-factor randomized complete block design and includes four treatments: CK2, B1, B2 and B3. The treatments involved foliar spraying per plant with 250 mL of water (CK2), 0.2 mg·L− 1 EBR solution (B1), 0.2 mg·L− 1 EBR mixed with a low-concentration solution (0.1 g·L− 1 α-Cyclodextrin, 0.1 mg·L− 1 Tween 80, 1 g·L− 1 CaCl2, 1.5 g·L− 1 ZnSO4·7H2O, and 2.0 g·L− 1 KH2PO4) (B2), and 0.2 mg·L− 1 EBR mixed with a high-concentration solution (0.1 g·L− 1 α-Cyclodextrin, 0.1 mg·L− 1 Tween 80, 2.0 g·L− 1 CaCl2, 3.0 g·L− 1 ZnSO4·7H2O, and 4.0 g·L− 1 KH2PO4) (B3). The experiment was conducted in 2022 following the same planting and field management protocols as previously described. On July 31, when the first central flower of tobacco plants reached 50% bloom, uniformly grown plants were topped to leave 20 leaves per plant, and foliar spraying was applied as described. After treatment for 10, 20, 30, 40, and 50 d, six representative plants were sampled from each treatment group. Three plants were used to measure chlorophyll content, and photosynthesis of the middle leaves (12th leaf position) and upper leaves (18th leaf position). Fresh leaf tissue (approximately 10.0 g) was collected from the middle of the upper leaves, 2.0 cm from each side of the main vein, using scissors. The samples were immediately placed in liquid nitrogen for gene expression analysis of BR biosynthesis and signal transduction, cold-related and senescence-related genes. The remaining three plants were assessed for agronomic traits, and then gently uprooted with a shovel, and about 10.0 g of fibrous roots were collected, cleaned with deionized water, and analyzed for root activity. Roots, stems, and leaves were separated, weighed, and oven-dried for dry matter accumulation. De-enzyme torrefaction was used to analyze conventional chemical components and nicotine accumulation in the upper leaves. Additionally, 1.0 kg of cured middle leaves (C3F) and upper leaves (B2F) from each treatment were analyzed for chemical composition.

Methods of measurement

Whether conditions

Weather data for the experimental site were obtained from the Henan provincial meteorological data center.

Agronomic traits

Agronomic traits were measured according to the “Investigating and measuring methods of agronomical character of tobacco” [49]. In each plot, five uniformly growing plants were selected to measure plant height, stem circumference, internode length, maximum leaf length, and maximum leaf width. The leaf area index (LAI) for each treatment was calculated using the formula: LAI = Σ(leaf length × leaf width × 0.6345) ÷ average ground area.

Photosynthetic capacity

Photosynthetic characteristics were measured between 9:00 and 11:00 AM using a photosynthesis meter (LI-6400, LI-COR, USA) under natural light conditions. The measurements included the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of the middle and upper leaves. The light intensity was set at 1200 µmol·m− 2·s− 1 in an open gas exchange system.

Root traits

To measure fresh root weight, roots were first cleaned with water, dried with absorbent paper, and then weighed. For dry root weight, roots were initially subjected to de-enzyme torrefaction in an oven at 105.0 °C for 30 min, then further dried at 60.0 °C until they reach a constant weight before being weighed. Root volume was determined using the water displacement method, and root activity was assessed using the reagent kit (Keming, Suzhou).

Dry matter accumulation

Different parts of the tobacco plant were washed thoroughly with clean water, dried with absorbent paper, and then cut with clean scissors. The roots, stems, and leaves were subjected to de-enzyme torrefaction in an oven at 105.0 °C for 30 min, followed by additional drying at 60.0 °C until a constant weight was achieved. The dried samples were then weighed.

Economic trait

Fifty normal tobacco plants were selected from each plot for yield recording. After curing, the tobacco leaves were graded according to the national 42-grade standard and evaluated based on the local standard purchase prices. The proportions of each grade of cured tobacco were calculated for each treatment. Yield, production value, average price, and the proportions of high-grade and medium-grade tobacco were determined for each plot.

Chemical composition

A portion of the flu-cured tobacco was dehydrated and dried in an oven at 60.0 °C for 7 h. For measurement of chemical components in tobacco samples after de-enzyme torrefaction, various parts of the tobacco plant were initially dried in an oven at 105.0 °C for 30 min, followed by dehydration and drying at 60.0 °C. After grinding, these samples were sieved through a 60-mesh sieve. The continuous flow chemical analyzer (AA3, Seal Analytical, Germany) was used to measure the conventional chemical components including total nitrogen, nicotine, total sugars, reducing sugars, potassium, and chlorine.

Chlorophyll content

The chlorophyll content in fresh tobacco leaves was determined using spectrophotometry.

Gene expression analysis

Real-time quantitative RT-PCR was performed to validate the expression of the candidate gene, following previously described methods [50]. The L25 gene served as an internal control. The primers used for qRT-PCR are listed in Supplementary Table S1.

Statistical analysis

The experimental data were organized and plotted using GraphPad Prism 8.0.1 (San Diego, USA). Data obtained were analyzed with one-way analysis of variance (ANOVA) using the Statistical Product Service Solutions (SPSS) software (v. 26.0, Chicago, IL, USA), and significance was tested using Duncan’s test, P < 0.05 was considered as statistically significant. The experiments described above were conducted for at least three biological replicates for each sample, and data are presented as the mean ± SD.

Results

Weather conditions of the experimental site

Flue-cured tobacco is a thermophilic crop, with the optimal growth temperature ranging from 22 to 28℃, and and temperatures should not fall below 20℃ during the maturation period [26]. The weather conditions at the experimental site for 2021–2022 are illustrated in Fig. 1. In 2021, the average temperature from the root spreading stage (S2) to the day of topping was 22.1℃, while from the day of topping to the end of the maturation, it averaged 20.1℃. During the maturation period (S5), the average temperature was 19.4℃ (Fig. 1A). In 2022, during the root spreading stage (S2), the minimum temperature for tobacco plants was 14.0℃ with an average of 22.5℃. In the early stage of the fast growing period (S3), temperatures were high, reaching a maximum of 39.0℃ with an average of 25.9℃. The mature stage (S5) experienced lower temperatures with an average of 19.6℃ and a minimum of 8.3℃ (Fig. 1B). These results indicated that the temperature of the maturation period was below the the optimal range in the Sanmenxia Lingbao tobacco region.

Fig. 1
figure 1

The weather conditions of flue-cured tobacco growth period in 2021 and 2022. S1 indicates the period from tobacco seedling transplanting to establishment, known as the seedling restitution stage. S2 indicates the period from seedling establishment to the rosette stage, known as the root spreading stage. S3 indicates the period from rosette stage to flower-bud appearing stage stage, known as the fast growing period. S4 indicates the period from flower-bud appearing stage to the rounded bud stage, known as the top leaf expansion stage. S5 indicates the period from 10 d after topping the tobacco leaves until the final harvest of the upper leaves, known as the mature stage. Red line indicates the day of treatment, also the day of topping the tobacco leaves

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Effects of different EBR concentrations on leaf nicotine content at maturity in low-temperature regions

The optimal application concentration of EBR varies among different crops [51, 52]. In tobacco plants, topping stimulates root redevelopment and the synthesis and accumulation of nicotine, significantly increasing nicotine levels post-topping [53, 54]. Therefore, different EBR concentrations were applied on the day of topping to determine the optimal concentration by comparing the agronomic and economic traits of flue-cured tobacco and the nicotine content in cured leaves.

We initially assessed the agronomic traits of tobacco plants treated with different concentrations of EBR. On the treatment day, there were no notable differences in these traits across the different EBR treatments. However, after 15 d, plants in the A1, A2, and A3 treatments exhibited significantly greater heights compared to the CK1 group. Among these, the A2 treatment demonstrated the most pronounced increases in stem girth, node distance, and maximum leaf length. After 30 d of treatment, all measured traits including plant height, stem girth, node distance, maximum leaf length, and leaf area index were significantly higher in EBR-treated plants, with the A2 treatment showing the most substantial improvements (Figure S1). Additionally, foliar application of EBR notably enhanced the yield, quality, and proportion of high- to medium-grade tobacco. Specifically, the A2 treatment yielded the highest values in yield, quality, average price, and proportion of high- to medium-grade tobacco (Table 1). As shown in Table 2, the middle and upper cured tobacco leaves in the A2 treatment exhibited the highest contents of total nitrogen, nicotine, total sugars, reducing sugars, and potassium, significantly surpassing the other treatments. Specifically, the total nitrogen content in these leaves increased by 14.39% and 26.99% in the middle and upper leaves, respectively, compared to CK1. Nicotine content also rose by 11.11% and 19.90% in the middle and upper leaves, respectively, compared to CK1. Chlorine content in the middle and upper cured leaves of A2 and A1 was significantly lower than in CK1 and A3.

Table 1 Foliar application of different concentrations of EBR significantly improves the economic properties of flue-cured tobacco
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Table 2 Foliar application of different concentrations of EBR significantly improves the chemical composition of cured tobacco leaves
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These findings indicate that foliar application of EBR on the day of topping effectively promotes the agronomic and economic traits of flue-cured tobacco, as well as leaf nicotine content, with the A2 treatment (0.2 mg·L− 1 EBR solution, 0.05 mg per plant) being the most advantageous.

The regulatory effects and mechanism of EBR compounds on leaf nicotine content during mature stage

Flue-cured tobacco, a warm-season crop, often encounters delayed leaf harvesting and reduced leaf quality in climates with low temperatures and heavy rainfall during its late growth stages. Cyclodextrin and Tween can regulate the release rate of pesticides and enhance their stability [39, 40]. CaCl2 and ZnSO4 help mitigate plant damage and improve cold tolerance under low temperature conditions [41, 42]. Additionally, KH2PO4 promotes yellowing and maturation of upper tobacco leaves [45,46,47]. Given these factors, it is crucial to explore the potential synergies between EBR and α-Cyclodextrin, Tween 80, CaCl2, ZnSO4·7H2O and KH2PO4 to enhance the leaf nicotine content.

Effects of applying different EBR compounds on the growth and development of tobacco plants

As shown in Figure S2, tobacco plants treated with B2 and B3 exhibited significantly higher plant height, stem girth, node distance, maximum leaf length, leaf width, and area index of the six upper leaves compared to CK2 from 10 to 50 d after treatment.

The net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) of the middle leaves decreased over time across all treatments (Figure S3A-3 C). However, 10 d after treatment, B3 had significantly higher Pn, Tr, and Gs compared to B1 and CK2. From 20 to 50 d post-treatment, the middle leaves in B2 and B3 maintained higher Pn, Tr, and Gs than those in B1 and CK2 (Figure S3A-3 C). Additionally, the intercellular CO2 concentration (Ci) in the middle leaves gradually increased among the treatments (Figure S3D). From 10 to 40 d post-treatment, Ci in CK2 was significantly higher than in the other treatments (Figure S3D). By 50 d post-treatment, there was no significant difference between CK2 and B1, both of which were higher than B2 and B3 (Figure S3D).

The upper leaves exhibited an initial increase followed by a decrease in Pn, Tr, and Gs across all treatments, reaching peak values 20 d after treatment (Figure S4A-4 C). From 20 to 50 d post-treatment, B3 showed the highest Pn, Tr, and Gs, significantly greater than B1 and CK2, with B2 following (Figure S4A-4 C). Ci in B1, B2, and B3 initially decreased and then increased, while CK2 showed a gradual rise (Figure S4D). After 20 d, B3 had the highest Ci (Figure S4D). Between 30 and 50 d post-treatment, B2 and B3 were not significantly different, both lower than B1 and CK2 (Figure S4D).

From 10 d to 50 d post-treatment, root volume, fresh weight, and dry weight exhibited an upward trend, while root activity showed a downward trend (Figure S5). These parameters rose rapidly in the first 30 d after treatment, then slowed (Figure S5A-5 C). In CK2, the physiological characteristics of root system were consistently the lowest across all periods, significantly lower than those in other treatments (Figure S5). Conversely, B3 treatment resulted in the highest root volume, fresh weight, dry weight, and root activity at each period post-treatment (Figure S5).

As shown in Figure S6, from 10 to 30 d post-treatment, dry matter accumulation increased rapidly across all treatments, then slowed from 30 to 50 d. After 10 d, dry matter accumulation in B1, B2, and B3 treatments was significantly higher than in CK2 (Figure S6). After 20 d, dry matter accumulation in the stem, middle leaves, upper leaves, and total per plant of B3 was significantly higher than in the other treatments, while CK2 had the lowest values (Figure S6). At 50 d post-treatment, Compared to CK2, B1, B2, and B3 treatments increased dry matter accumulation in the stem by 39.59%, 49.54%, and 60.63%, respectively, and in the upper leaves by 31.58%, 41.27%, and 51.55%, respectively (Figure S6A).

As shown in Table 3, the economic indicators (yield, output value, proportion of high-quality tobacco, and proportion of medium to high-quality tobacco) in B3 were higher than in the other treatments, followed by B2. Specifically, the yield for B1, B2, and B3 treatments increased by 25.49%, 31.23%, and 36.19%, respectively, compared to CK2. Output values for B1, B2, and B3 was 54374.55 RMB·ha− 1, 57166.35 RMB·ha− 1, and 60726.30 RMB·ha− 1 higher than that CK2. The average price of B3 treatment exceeded 27.00 RMB·kg− 1, and the proportion of high-quality tobacco in B3 treatment exceeded 50%.

Table 3 Foliar application of different EBR compounds significantly improves economic characters of flue-cured tobacco
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The above results indicate that spraying EBR compounds enhance agronomic and economic traits of flue-cured tobacco, with the B3 treatment demonstrating the most pronounced effect.

Effects of applying different EBR compounds on the content of chemical components in upper tobacco leaves

Over time, total nitrogen content in upper tobacco leaves gradually decreased, while nicotine content gradually increased (Table 4). Total sugar and reducing sugar levels initially rose and then declined. Potassium content steadily decreased, whereas chloride content first decreased and then increased. Throughout the entire period, total sugar and reducing sugar contents were significantly higher under B2 and B3 treatments compared to B1 and CK2. And total nitrogen content in upper leaves was significantly higher under B1, B2, and B3 treatments compared to CK2. From 30 to 50 d post-treatment, the B3 treatment had the highest total nitrogen content. Specifically, at 50 d, total nitrogen content increased by 14.87%, 25.64%, and 27.69% under B1, B2, and B3 treatments, respectively, compared to CK2. Nicotine content in upper leaves was similar for B2 and B3 between 10 and 30 d, both significantly higher than B1 and CK2. From 40 to 50 d post-treatment, nicotine content showed significant differences among all treatments, with B3 having the highest content, followed by B2, B1, and CK2. At 50 d, nicotine content increased by 13.48%, 21.91%, and 28.09% under B1, B2, and B3 treatments, respectively, compared to CK2. Significant differences in potassium content were observed among treatments, except at 30 d, with the order being B3 > B2 > B1 > CK2. Chlorine content was consistently lower in the upper leaves of B1 compared to other treatments. In B2, chlorine content was significantly lower at 10 and 20 d post-treatment, and in B3, at 10 and 30 d post-treatment, compared to CK2. No significant differences were observed at other times compared to CK2.

Table 4 Foliar application of different EBR compounds significantly improves chemical components in upper tobacco leaves during its developmental stages
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We also measured the nicotine accumulation in the upper leaves over time (Fig. 2). Nicotine accumulation increased gradually, peaking between 20 and 30 d after treatment. Throughout the study period, nicotine accumulation was significantly higher in B3 and B2 treatments compared to B1 and CK2. Notably, at 20, 40, and 50 d post-treatment, nicotine accumulation in B3 was significantly higher compared to all other treatments. At 50 d post-treatment, nicotine accumulation in the upper leaves for B1, B2, and B3 increased by 49.38%, 72.25%, and 94.73%, respectively, compared to CK2. Additionally, the content of chemical components in cured tobacco leaves was significantly higher in B1, B2, and B3 treatments than in CK2, except for chlorine content (Table 5). Furthermore, the B3 treatment showed the highest levels of total nitrogen, nicotine, total sugar, reducing sugar, and potassium in both the middle and upper cured tobacco leaves, with total nitrogen content increasing by 13.75% and 29.14% in the middle and upper leaves, respectively, compared to CK2. Nicotine content also rose by 24.14% and 29.00%.

Fig. 2
figure 2

Foliar application of different EBR compounds significantly enhances nicotine accumulation in the upper leaves of flue-cured tobacco during its developmental stages. CK2 means foliar spraying with 250 mL of water per plant; B1 indicates foliar spraying with 250 mL of 0.2 mg·L− 1 EBR solution per plant; B2 indicates foliar spraying of 250 mL of a mixed solution containing 0.2 mg·L− 1 EBR, 0.1 g·L− 1 α-Cyclodextrin, 0.1 mg·L− 1 Tween 80, 1 g·L− 1 CaCl2, 1.5 g·L− 1 ZnSO4·7H2O, and 2 g·L− 1 KH2PO4 per plant. B3 indicates foliar spraying of 250 mL of a mixed solution containing 0.2 mg·L− 1 EBR, 0.1 g·L− 1 α-Cyclodextrin, 0.1 mg·L− 1 Tween 80, 2 g·L− 1 CaCl2, 3 g·L− 1 ZnSO4·7H2O, and 4 g·L− 1 KH2PO4. 10 d, 20 d, 30 d, 40 d and 50 d indicate the days after treatment with different EBR compounds. The data presented are the mean ± SD. Different letters indicate significant differences among the treatments based on Duncan’s test (P < 0.05)

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Table 5 Foliar application of different EBR compounds significantly improves chemical composition of cured tobacco leaves
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These findings indicate that foliar application of the EBR compounds significantly enhance the total nitrogen, nicotine, total sugar, reducing sugar, and potassium content in both upper leaves and cured leaves, with the B3 treatment showing the most pronounced effects.

Mechanism of EBR compounds on nicotine content in mature tobacco leaves

BRs are known to enhance crop growth by regulating cell division and elongation [55,56,57,58]. We hypothesized that EBR compounds influence the expression of key genes involved in BR biosynthesis and signaling, specifically NtDWF4 (a BR biosynthesis gene), NtBZR1 and NtBZR2 (BR signaling genes), and NtHERK2 (a gene involved in BR-regulated cell expansion). Our results confirm this hypothesis. Foliar application of EBR (B1) and EBR compounds (B2 and B3) significantly enhanced the expression levels of NtDWF4, NtBZR1, NtBZR2, and NtHERK2 in the upper leaves compared to the CK2 treatment at various stages (Fig. 3).

Fig. 3
figure 3

Foliar application of different EBR compounds significantly increases the expression of genes involving in BR biosynthesis (NtDWF4), signal transduction (NtBZR1 and NtBZR2), BR-regulated cell expansion (NtHERK2) during the development of upper leaves. CK2, B1, B2 and B3 indicate as shown in Fig. 2. 10 d, 20 d, 30 d, 40 d and 50 d indicate as shown in Fig. 2. The data presented are the mean ± SD. Different letters indicate significant differences among the treatments based on Duncan’s test (P < 0.05)

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Chlorophyll content is crucial for the growth and yield of flue-cured tobacco, with reductions often signaling leaf senescence [59]. To understand how EBR compounds impact this, we measured chlorophyll content and the expression levels of senescence-related genes in the upper leaves. During the treatment period, chlorophyll content in the upper leaves initially increased and then decreased (Figure S7). The B2 treatment consistently maintained higher chlorophyll levels at all stages compared to B1 and CK2, with B3 following closely behind (Figure S7). Correspondingly, the expression levels of senescence-promoting genes, NtCP1 and NtCP23, were notably lower in the B1, B2, and B3 treatments compared to CK2 (Fig. 4A and B). Conversely, the expression levels of senescence-repressing genes, NtPSA1 and NtMC, were significantly higher in these treatments (Fig. 4C and D). After 30 d of treatment, NtCP1 and NtCP23 levels increased across all treatments, indicating significant senescence changes (Fig. 4A and B). By 40 and 50 d post-treatment, NtPSA1 and NtMC expression was significantly higher in B2 compared to other treatments (Fig. 4C and D), which is consistent with that B2 possessing the higher chlorophyll content (Figure S7).

Fig. 4
figure 4

Foliar application of different EBR compounds significantly represses the expression of senescence-promoting genes (NtCP1 and NtCP23) and promotes the expression of senescence-repressing genes (NtPSA1 and NtMC) during the development of upper leaves. CK2, B1, B2 and B3 indicate as shown in Fig. 2. 10 d, 20 d, 30 d, 40 d and 50 d indicate as shown in Fig. 2. The data presented are the mean ± SD. Different letters indicate significant differences among the treatments based on Duncan’s test (P < 0.05)

Full size image

Previous research has highlighted the crucial role of BRs in helping plants manage cold stress [34, 60]. A key player in this process is the C-repeat binding factor (CBF), which acts as a central transcription factor in the cold stress response regulatory network [61]. To evaluate how EBR compounds affect this response, we measured the relative expression levels of CBF genes (NtCBF1 and NtCBF2) and their target genes (NtCOR47 and NtCOR78) in the upper leaves at different stages following foliar application of EBR compounds. The results indicated that there were no significant differences in the expression levels of NtCBF1, NtCBF2, NtCOR47, and NtCOR78 among the treatments at 10 and 20 d post-treatment (Fig. 5). However, from 30 to 50 d post-treatment, the expression levels of NtCBF1 and NtCBF2 were notably higher in the B2 and B3 treatments compared to the other treatments (Fig. 5A and B). Similarly, B2 and B3 treatments showed significantly higher expression levels of NtCOR47 and NtCOR78 during this period (Fig. 5C and D).

Fig. 5
figure 5

Foliar application of different EBR compounds significantly enhances the expression of cold-related genes (NtCBF1, NtCBF2, NtCOR47, and NtCOR78) in upper leaves after treatment for 30, 40, and 50 d. CK2, B1, B2 and B3 indicate as shown in Fig. 2. 10 d, 20 d, 30 d, 40 d and 50 d indicate as shown in Fig. 2. The data presented are the mean ± SD. Different letters indicate significant differences among the treatments based on Duncan’s test (P < 0.05)

Full size image

These findings suggest that EBR compounds enhance the leaf nicotine content by promoting BR metabolism-related genes, senescence-repressing genes, and cold tolerance-related genes, while inhibiting the expression of senescence-promoting genes during mature stages.

Discussion

The broader implications for tobacco agriculture in different climates

Tobacco, a key economic crop, is valued primarily for its leaves. The tobacco industry significantly contributes to national revenue and gross domestic product (GDP), promotes local economic development, boosts the income of tobacco farmers, and exerts a considerable impact on the global economy [62]. However, tobacco cultivation is highly vulnerable to adverse agroclimatic conditions, and climate change has significantly impacted its production, presenting substantial challenges [63].

High temperatures adversely affect tobacco plants at various growth stages. During the seedling stage, temperatures above 30 °C cause leaf scorching, resulting in dark yellow and wrinkled foliage [64]. During the rosette and vigorous growth stages, prolonged heat leads to leaf wilting, deformities, and necrotic spots, potentially resulting in plant death under severe conditions [65]. At the late growth stage, high temperatures slow plant growth, disrupt leaf yellowing stratification, and may trigger premature ripening [26].

Similarly, cold stress negatively impacts tobacco plants. During the seedling stage, cold exposure causes leaf wilting and shrinkage, inhibiting growth, reducing plant height, and decreasing the number of effective leaves. It can also lead to early flowering [26]. Additionally, cold stress affects leaf quality by altering microstructure, chlorophyll content, and maturity, compromising curing characteristics and ultimately reducing the quality of cured leaves [27,28,29].

Drought is another critical limiting factor for tobacco growth. It significantly hampers root development during vigorous growth, reducing root indicators and slowing dry matter accumulation [66]. Severe water shortages at any stage lead to poor root growth, reduce allocation of dry matter to aboveground parts, and increase root-to-shoot ratios [67]. Adequate soil moisture is crucial for leaf development, as it positively correlates with leaf number and expansion. In contrast, insufficient moisture impairs leaf development by decreasing leaf count, causing premature aging of lower leaves, shrinking and thickening upper leaves, and roughening leaf tissue [68].

Together, these climatic stressors highlight the vulnerability of tobacco cultivation to environmental changes, underscoring the need for adaptive strategies to mitigate their impact.

Exogenous EBR promotes the growth and development of tobacco plants

Low temperatures during the maturation period hinder the growth of upper leaves, adversely affecting the yield and quality of flue-cured tobacco. BRs are known to improve crop resilience, improving both yield and quality [69, 70]. Our results demonstrate that exogenous EBR application significantly promotes the growth, development, and the overall yield of mature tobacco plants (Figure S1, S2 and Table 1), with 0.2 mg·L− 1 EBR showing the most pronounced effects. Higher EBR concentrations, while beneficial, exhibited diminishing returns in plant height, stem circumference, and leaf area, potentially due to EBR-induced inhibition of gibberellin (GA) synthesis, which may limit plant growth [71].

Studies have shown that BRs promote plant growth by regulating cell division and elongation [55,56,57,58]. DWF4 (CYP90), encoding a cytochrome P450 enzyme, plays a key role in BR biosynthesis and cell elongation [72,73,74]. BRs also activate target genes such as BZR1 and BES1 (BZR2), which enhance cell elongation [75, 76], while HERK2, regulated by BES1, contributes to this process [77]. Consistent with these mechanisms, foliar application of EBR (B1) significantly enhanced the expression levels of NtDWF4, NtBZR1, NtBZR2, and NtHERK2 in tobacco leaves compared to the CK2 treatment (Fig. 3).

Exogenous EBR enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco

Nitrogen (N) is a crucial macro-nutrient for plant growth, and efficient nitrogen absorption and assimilation are essential for improving nitrogen use efficiency [78, 79]. Studies show that BRs enhance nitrogen metabolism through multiple regulatory mechanisms. For example, BR signaling kinase BSK3 regulates root elongation under mild nitrate deficiency, and low N specifically enhances the expression of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation [80]. Mild low N also enhances the BR biosynthesis gene DWARF1 (DWF1), with natural variations in DWF1 contributing to the nitrogen foraging response [81]. Under low nitrogen conditions, BES1 interacts with LBD37 to modulate brassinosteroid-regulated root forging response in Arabidopisis [82]. In rice, BR might regulate the nitrogen-use efficiency by modulating the activities of OsTCP19 and DLT [83]. As a synthetic derivative of BRs, EBR plays a critical role in enhancing nitrogen absorption and assimilation under stress conditions. For instance, EBR mitigates nitrate flux reduction in cucumber roots [37], boosts nitrogen assimilation enzyme activity in salt-stressed chickpea [84], and regulates NRT gene expression in maize seedlings under low nitrate conditions [85]. However, these studies primarily focus on the seedling stage. Here, foliar application of EBR significantly increased total nitrogen and nicotine content in the upper leaves during the mature stage of flue-cured tobacco (Tables 2, 4 and 5; Fig. 2). Our prior research demonstrated that both foliar spraying and root drenching of EBR improved the activity of key nitrogen absorption and assimilation enzymes (NR, GS) in upper leaves, enhanced the expression of nitrogen assimilation-related genes (NtGS2, NtGDH1 and NtNR), nicotine synthesis genes (NtQPT, NtPMT), and nicotine transport genes (NtJAT1, NtJAT2 and NtNUP1) in various parts of plant tissues [86]. This led to substantial increases in total nitrogen and nicotine content in cured middle and upper leaves [86]. Consequently, these genes were not re-analyzed in this study.

Applying exogenous EBR boosts the nicotine content in tobacco leaves without additional nitrogen input (Tables 2, 4 and 5; Fig. 2). This effect likely results from EBR’s capacity to regulate nitrogen metabolism during tobacco maturation, and enhance cold resistance [34, 60]. During the 2021 and 2022 maturation periods, the average daily temperature remained below 20.0 °C (Fig. 1). In 2022, temperatures dropped sharply 30 d after applying EBR, with the daily average falling to just 14.8 °C within 4 d. Under these conditions, EBR-treated plants (B1) showed significantly higher expression levels of cold-response genes NtCBF1, NtCBF2, NtCOR47, and NtCOR78 in the upper leaves after 30 d compared to CK2 (Fig. 5). This aligns with previous findings that exogenous BRs enhance cold tolerance in Arabidopsis by up-regulating CBF1 and COR47 expression under cold stress [61].

Premature or delayed senescence in the upper leaves of flue-cured tobacco negatively impacts the accumulation of essential quality compounds, ultimately reducing leaf quality [87]. In tobacco, senescence is marked by reduced chlorophyll content, with advanced senescence leading to significant chlorophyll degradation. This study found that EBR-treated leaves (B1) retained higher chlorophyll levels than CK2 at all stages post-treatment (Figure S7). Additionally, senescence-promoting genes NtCP1 and NtCP23 were expressed at significantly lower levels, while senescence-inhibiting genes NtPSA1 and NtMC were expressed at significantly higher levels in B1-treated plants compared to CK2 (Fig. 4). These results suggest that foliar application of EBR delays senescence, promoting better growth and development, and ultimately enhancing leaf nicotine content.

The EBR compounds enhance the effectiveness of exogenous EBR application

The EBR compounds comprises α-Cyclodextrin, Tween 80, CaCl2, ZnSO4·7H2O, and KH2PO4. Their application significantly improved agronomic and economic traits, as well as the total nitrogen, nicotine, and potassium content in tobacco leaves, with the B3 treatment yielding the most pronounced effects (Fig. 2, Figure S2-S6, Tables 4 and 5). This enhanced performance is likely due to the α-Cyclodextrin and Tween 80 regulating EBR release, improving its stability, viscosity, and adherence to leaf surfaces, thereby extending its efficacy [39, 40]. As a result, photosynthetic capacity, root development, and leaf growth were markedly improved, leading to higher nicotine levels (Fig. 2, Figure S2-S5, Tables 4 and 5). Additionally, leaves treated with EBR compounds exhibited significantly higher potassium levels compared to those treated with EBR alone, likely due to the absorption of KH2PO4 from the formulation (Tables 4 and 5).

After treatment for 30 d, the expression levels of NtCBF1 and NtCBF2, along with their target genes NtCOR47 and NtCO78, were significantly higher in the upper leaves of plants treated with the B2 and B3 (EBR compounds) compared to those treated with B1 (single EBR) (Fig. 5). This may be due to CaCl2 and ZnSO4·7H2O in the compounds enhancing the cold resistance of tobacco plants [41, 42]. From 10 to 50 d post-treatment, chlorophyll content in the upper leaves was significantly higher in B2-treated plants than in B1-treated plants (Figure S7). Similarly, B3-treated plants showed higher chlorophyll content than B1-treated plants from 10 to 40 d post-treatment (Figure S7). This increase in chlorophyll content may be attributed to the α-Cyclodextrin and Tween 80 in the compounds, which extend EBR’s effectiveness and delay leaf senescence. However, by 50 d post-treatment, no significant differences in chlorophyll content were observed between B3 and B1 treatments, both of which were significantly lower than B2 (Figure S7). Correspondingly, the expression levels of senescence-inhibiting marker genes NtPSA1 and NtMC in the upper leaves did not differ significantly between B1 and B3 treatments but were significantly lower compared to B2 (Fig. 4C and D). This may be due to the higher concentration of KH2PO4 in the B3 compound, which accelerates plant maturity and senescence.

Conclusions

This study found that foliar application of EBR at different concentrations (0.1, 0.2 and 0.4 mg·L− 1) influenced growth, economic traits, and chemical compounds, including leaf nicotine content, during the mature stage of flue-cured tobacco. The optimal concentration was 0.2 mg·L− 1. Furthermore, foliar application of different EBR compounds enhances cold tolerance, promotes plant growth, improves root activity, and boosts total nitrogen and nicotine content in the upper leaves. Notably, B3 treatment (0.2 mg·L− 1 EBR with 0.1 g·L− 1 α-Cyclodextrin, 0.1 mg·L− 1 Tween 80, 2.0 g·L− 1 CaCl2, 3.0 g·L− 1 ZnSO4·7H2O, and 4.0 g·L− 1 KH2PO4) significantly enhances the nicotine content in middle and upper cured leaves by 9.64% and 12.03%, respectively, compared to the single EBR treatment.

Applying EBR without increasing nitrogen at the mature stage enhances the cold resistance, nitrogen, and nicotine metabolism of tobacco plants, increasing nicotine content in the leaves. This approach reduces growers’ costs, boosts income, and aids policymakers in developing standards to support agricultural growth. However, it should be be considered potential risks such as water pollution, changes in soil microbial communities, and interactions with other pesticides, which may affect crop health and environmental quality.

Data availability

The data presented in this study are available upon request from the corresponding author.

References

  1. Moghbel N, Ryu B, Ratsch A, Steadman KJ. Nicotine alkaloid levels, and nicotine to nornicotine conversion, in Australian Nicotiana species used as chewing tobacco. Heliyon. 2017;3(11):e00469.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Baldwin IT. The alkaloidal responses of wild tobacco to real and simulated herbivory. Oecologia. 1988;77:378–81.

    Article  PubMed  Google Scholar 

  3. Yoshida D, Takahashi T. Relation between the behaviour of nitrogen and the nicotine synthesis in tobacco plant. Soil Sci Plant Nutr. 1961;7:157–64.

    Article  Google Scholar 

  4. Patel H, McIntire J, Ryan S, Dunah A, Loring R. Anti-inflammatory effects of astroglial α7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-κB pathway and activation of the Nrf2 pathway. J Neuroinflammation. 2017;14(1):192.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Leino S, Koski SK, Rannanpää S, Salminen O. Effects of antidyskinetic nicotine treatment on dopamine release in dorsal and ventral striatum. Neurosci Lett. 2018;672:40–5.

    Article  CAS  PubMed  Google Scholar 

  6. Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT. Nicotine’s defensive function in nature. PLoS Biol. 2004;2(8):E217.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Li Z, Li Z, Liu Z, Wang Z, Lei B, Li J, et al. Joint insecticidal action of farnesol and nicotine against Aphis citricolavander goot. Chin J Biol Ctrl. 2019;35(1):37–43.

    CAS  Google Scholar 

  8. Mukhopadhyay I, Chowdhuri DK, Bajpayee M, Dhawan A. Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline Comet assay. Mutagenesis. 2004;19(2):85–90.

    Article  CAS  PubMed  Google Scholar 

  9. Moore EL, Gwynn GR, Powell NT. Some alkaloid relationships among seedings and mature plants of FCT. Tob Sci. 1963;7:170–8.

    CAS  Google Scholar 

  10. Legg PD, Chaplin JF, Collins GB. Inheritance of percent total alkaloids in Nicotiana tabacum L. populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. J Hered. 1969;60:213–7.

    Article  Google Scholar 

  11. Legg PD, Collins GB. Inheritance of percent total alkaloids in Nicotiana tabacum L. II. Genetic effects of two loci in Burley 21 LA Burley 21 populations. Can J Genet Cytol. 1971;13:287–91.

    Article  Google Scholar 

  12. Crockford RH. Effect of amount and time of application of nitrogen on the nicotine content of tobacco leaves. Aust J Exp Agr. 1977;17:469–74.

    Article  CAS  Google Scholar 

  13. Williams LM, Miner GS. Effect of urea on yield and quality of flue-cured tobacco. Agron J. 1982;74:457–62.

    Article  Google Scholar 

  14. Walch-Liu P, Neumann G, Bangerth F, Engels C. Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J Exp Bot. 2000;51(343):227–37.

    Article  CAS  PubMed  Google Scholar 

  15. Garrido-Lestache E, López-Bellido RJ, López-Bellido L. Effect of N rate, timing and splitting and N type on bread-making quality in hard red spring wheat under rainfed Mediterranean conditions. Field Crops Res. 2004;85:213–36.

    Article  Google Scholar 

  16. Tang J, Chun C, Jiang L, Feng J, Christire P, Suo Z. Yield and nicotine content of flue-cured tobacco as affected by soil nitrogen mineralization. Pedosphere. 2008;18(2):227–35.

    Article  Google Scholar 

  17. Saunders JW, Bush LP. Nicotine biosynthetic enzyme activities in Nicotiana tabacum L. genotypes with different alkaloid levels. Plant Physiol. 1979;64(2):236–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cane KA, Mayer M, Lidgett AJ, Michael AJ, Hamill JD. Molecular analysis of alkaloid metabolism in AABB v. aabb genotype Nicotiana tabacum in response to wounding of aerial tissues and methyl jasmonate treatment of cultured roots. Funct Plant Biol. 2005;32:305–20.

    Article  CAS  PubMed  Google Scholar 

  19. Kartusch R, Mittendorfer B. Ultraviolet radiation increases nicotine production in Nicotiana callus cultures. J Plant Physiol. 1990;136(1):110–4.

    Article  CAS  Google Scholar 

  20. Cheng T, Hu L, Wang P, Yang X, Peng Y, Lu Y, et al. Carbon monoxide potentiates high temperature-induced nicotine biosynthesis in tobacco. Int J Mol Sci. 2018;19(1):188.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Jiang F. Research on the regulation techniques for nicotine accumulation and distribution in flue-cured tobacco. Doctoral dissertation, HUNAU, Changsha. 2007.

  22. Sanghera GS, Wani SH, Hussain W, Singh NB. Engineering cold stress tolerance in crop plants. Curr Genom. 2011;12(1):30–43.

    Article  CAS  Google Scholar 

  23. Pareek A, Khurana A, Sharma AK, Kumar R. An overview of signaling regulons during cold stress tolerance in plants. Curr Genom. 2017;18(6):498–511.

    Article  CAS  Google Scholar 

  24. Adhikari L, Baral R, Paudel D, Min D, Makaju SO, Poudel HP, et al. Cold stress in plants: strategies to improve cold tolerance in forage species. Plant Stress. 2022;4:100081.

    Article  CAS  Google Scholar 

  25. Zhang W, Huang W, Yang Q, Zhang S, Hu H. Effect of growth temperature on the electron flow for photorespiration in leaves of tobacco grown in the field. Physiol Plant. 2013;149(1):141–50.

    Article  CAS  PubMed  Google Scholar 

  26. Li Y, Ren K, Hu M, He X, Gu K, Hu B, et al. Cold stress in the harvest period: effects on tobacco leaf quality and curing characteristics. BMC Plant Biol. 2021;21(1):131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Meng K, Nie R, Xiao C, Tang C. Changes of pigment and moisture content in cured tobacco leaves during bulk curing process. J Hunan Agric Univ (Nat Sci). 2006;2:144–8.

    Google Scholar 

  28. Wang C, Zhang Z, Xu X, Lan Z, Gao Y, Qiu Q, et al. Advance in leaf curing characteristics of flue-cured tobacco. Chin Tob Sci. 2009;30(1):38–41.

    Google Scholar 

  29. Wang Y, Lu X, Yang X, Zhang S, Zhang J. Preliminary report on the flue-curing characteristics of Yunnan202, a new flue-cured tobacco variety. Chin Agric Sci Bull. 2007;37(5):662–9.

    Google Scholar 

  30. Ali B. Brassinosteroids: the promising plant growth regulators in horticulture. Brassinosteroids: Plant Growth Dev. 2019:349–65.

  31. Yadav RK, Devi LL, Singh AP. Brassinosteroids in plant growth and development [M]//Plant hormones in crop improvement. Elsevier. 2023.pp. 185–203.

  32. Cheon J, Fujioka S, Dilkes BP, Choe S. Brassinosteroids regulate plant growth through distinct signaling pathways in Selaginella and Arabidopsis. PLoS ONE. 2013;8(12):e81938.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Xia X, Huang L, Zhou Y, Mao W, Shi K, Wu J, et al. Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus. Planta. 2009;230:1185–96.

    Article  CAS  PubMed  Google Scholar 

  34. Singh I, Kumar U, Singh SK, Gupta C, Singh M, Kushwaha SR. Physiological and biochemical effect of 24-epibrassinoslide on cold tolerance in maize seedlings. Physiol Mol Biol Plants. 2012;18(3):229–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Behnamnia M, Kalantari M, Rezanejad F. Exogenous application of brassinosteroid alleviates drought-induced oxidative stress in Lycopersicon esculentum L. Gen Appl Plant Physiol. 2009;35:22–34.

    CAS  Google Scholar 

  36. Yu B, Wang L, Guan Q, Xue X, Gao W, Nie P. Exogenous 24-epibrassinolide promoted growth and nitrogen absorption and assimilation efficiency of apple seedlings under salt stress. Front Plant Sci. 2023;14:1178085.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Anwar A, Li Y, He C, Yu X. 24-Epibrassinolide promotes NO3– and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature. BMC Plant Biol. 2019;19(1):225.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Shu S, Tang Y, Yuan Y, Sun J, Zhong M, Guo S. The role of 24-epibrassinolide in the regulation of photosynthetic characteristics and nitrogen metabolism of tomato seedlings under a combined low temperature and weak light stress. Plant Physiol Biochem. 2016;107:344–53.

    Article  CAS  PubMed  Google Scholar 

  39. Villaverde J, Pérez-Martínez JI, Maqueda C, Ginés JM, Morillo E. Inclusion complexes of alpha- and gamma-cyclodextrins and the herbicide norflurazon: I. Preparation and characterisation. II. Enhanced solubilisation and removal from soils. Chemosphere. 2005;60(5):656–64.

    Article  CAS  PubMed  Google Scholar 

  40. Zhang A, Liu W, Wang L, Wen Y. Characterization of inclusion complexation between fenoxaprop-p-ethyl and cyclodextrin. J Agric Food Chem. 2005;53(18):7193–7.

    Article  CAS  PubMed  Google Scholar 

  41. Shi H, Ye T, Zhong B, Liu X, Chan Z. Comparative proteomic and metabolomic analyses reveal mechanisms of improved cold stress tolerance in bermudagrass (Cynodon dactylon (L.) Pers.) By exogenous calcium. J Integr Plant Biol. 2014;56(11):1064–79.

    Article  CAS  PubMed  Google Scholar 

  42. Liu Z, Meng J, Sun Z, Su J, Luo X, Song J, et al. Zinc application after low temperature stress promoted rice tillers recovery: aspects of nutrient absorption and plant hormone regulation. Plant Sci. 2022;314:111104.

    Article  CAS  PubMed  Google Scholar 

  43. Li J, Li Z, Li X, Tang X, Liu H, Li J, et al. Effects of spraying KH2PO4 on flag leaf physiological characteristics and grain yield and quality under heat stress during the filling period in winter wheat. Plants (Basel). 2023;12(9):1801.

    CAS  PubMed  Google Scholar 

  44. Yang J, Duan L, He H, Li Y, Li X, Liu D, et al. Application of exogenous KH2PO4 and salicylic acid and optimization of the sowing date enhance rice yield under high-temperature conditions. J Plant Growth Regul. 2022;41:1532–46.

    Article  CAS  Google Scholar 

  45. Jing Y, Yuan X, Wang W, Sun M, Liu Y, Lu P et al. Effects of spraying potassic fertilizers after topping on chemical composition of flue-cured tobacco. Hunan Agric Sci. 2015;(10):51–4.

  46. Yang Y, Zhang J, Dong S, Ye C, Hu L, Lv K, et al. Effect of spraying potassium fertilizer on the yield and quality of over- green and spindling flue-cured tobacco after topping. Guizhou Agric Sci. 2019;47:28–32.

    CAS  Google Scholar 

  47. Dong K, Mao J, Kong D, Zhang X, Zhao H, He L, et al. Effects of foliar spraying of different regulators on quality of tobacco leaves. Hubei Agric Sci. 2020;59:108–111116.

    Google Scholar 

  48. Li Y, Wang Y, Ma W, Tan C. Breeding and selecting of a new flue-cured tobacco variety Yunyan 87 and its characteristics. Chin Tob Sci. 2001;(4):38–42.

  49. YC/T 142–2010. Investigating and measuring methods of agronomical character of tobacco. Standards Press of China. 2010.

  50. Zhang X, Li J, Xing X, Li H, Zhang S, Chang J, et al. Transcriptome disclosure of hormones inducing stigma exsertion in Nicotiana tabacum by corolla shortening. BMC Genomics. 2024;25(2):320.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Dou L, Sun Y, Li S, Ge C, Shen Q, Li H, et al. Transcriptomic analyses show that 24-epibrassinolide (EBR) promotes cold tolerance in cotton seedlings. PLoS ONE. 2021;16(2):e0245070.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Liu X, Zhu Q, Liu W, Zhang J. 24-Epibrassinolide confers zinc stress tolerance in watermelon seedlings through modulating antioxidative capacities and lignin accumulation. PeerJ. 2023;11:e15330.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Fan J, Yang J, Wang S, Xiao Z, Liang M, Li H, et al. Effect of topping on growth of flue-cured tobacco and nicotine accumulation and distribution in different organs. Crop Res. 2016;30:36–40.

    Google Scholar 

  54. Yang H, Zhou J, Luo Z, Wang Y, Zhou W. Influence of topping on the chemical components in the flue-cured tobacco during its different stages. Hunan Agric Sci. 2004;04:19–22.

    Google Scholar 

  55. Heyman J, Cools T, Vandenbussche F, Heyndrickx KS, Van Leene J, Vercauteren I, et al. ERF115 controls root quiescent center cell division and stem cell replenishment. Science. 2013;342(6160):860–3.

    Article  CAS  PubMed  Google Scholar 

  56. Jin H, Do J, Shin SJ, Choi JW, Choi YI, Kim W, et al. Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem of Liriodendron tulipifera. Phytochemistry. 2014;101:40–51.

    Article  CAS  PubMed  Google Scholar 

  57. Kang YH, Breda A, Hardtke CS. Brassinosteroid signaling directs formative cell divisions and protophloem differentiation in Arabidopsis root meristems. Development. 2017;144(2):272–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Vogler F, Schmalzl C, Englhart M, Bircheneder M, Sprunck S. Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod. 2014;27(3):153–67.

    Article  CAS  PubMed  Google Scholar 

  59. Guo Y, Ren G, Zhang K, Li Z, Miao Y, Guo H. Leaf senescence: progression, regulation, and application. Mol Hortic. 2021;1(1):5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Anwar A, Bai L, Miao L, Liu Y, Li S, Yu X, et al. 24-Epibrassinolide ameliorates endogenous hormone levels to enhance low-temperature stress tolerance in cucumber seedlings. Int J Mol Sci. 2018;19(9):2497.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science. 1998;280(5360):104–6.

    Article  CAS  PubMed  Google Scholar 

  62. Liu M, Liu X, Song Y, Hu Y, Yang C, Li J, et al. Tobacco production under global climate change: combined effects of heat and drought stress and coping strategies. Front Plant Sci. 2024;15:1489993.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Fahad S, Bajwa AA, Nazir U, Anjum SA, Farooq A, Zohaib A, et al. Crop production under drought and heat stress: plant responses and management options. Front Plant Sci. 2017;8:1147.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Pearce RC, Bailey WA, Bush LP, Green JD, Jack AM, Miller RD et al. 2015–2016 Burley and Dark Tobacco Production Guide. 2015. Available onlineat:https://smith.tennessee.edu/wp-content/uploads/sites/209/2020/11/TobaccoProduction-Guide.pdf

  65. Bittner RJ, Arellano C, Mila AL. Effect of temperature and resistance of tobacco cultivars to the progression of bacterial wilt, caused by Ralstonia solanacearum. Plant Soil. 2016;408:299–310.

    Article  CAS  Google Scholar 

  66. Kumar J, Sen Gupta D, Djalovic I, Kumar S, Siddique KHM. Rootomics for drought tolerance in cool-season grain legumes. Physiol Plant. 2021;172:629–44.

    Article  CAS  PubMed  Google Scholar 

  67. Martiniello P, Teixeira da Silva JA. Physiological and bioagronomical aspects involved in growth and yield components of cultivated forage species in Mediterranean environments: a review. Eur J Plant Sci Biotechnol. 2011;5:64–98.

    Google Scholar 

  68. Tang Z, Chen L, Chen Z, Fu Y, Sun X, Wang B, et al. Climatic factors determine the yield and quality of Honghe flue-cured tobacco. Sci Rep. 2020;10(1):19868.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Anjum SA, Wang L, Farooq M, Hussain M, Xue L, Zou C. Brassinolide application improves the drought tolerance in maize through modulation of enzymatic antioxidants and leaf gas exchange. J Agron Crop Sci. 2011;197(3):177–85.

    Article  CAS  Google Scholar 

  70. Divi UK, Rahman T, Krishna P. Gene expression and functional analyses in brassinosteroid-mediated stress tolerance. Plant Biotechnol J. 2016;14(1):419–32.

    Article  CAS  PubMed  Google Scholar 

  71. Tong H, Xiao Y, Liu D, Gao S, Liu L, Yin Y, et al. Brassinosteroid regulates cell elongation by modulating gibberellin metabolism in rice. Plant Cell. 2014;26(11):4376–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Szekeres M, Németh K, Koncz-Kálmán Z, Mathur J, Kauschmann A, Altmann T, et al. Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell. 1996;85(2):171–82.

    Article  CAS  PubMed  Google Scholar 

  73. Choe S, Dilkes BP, Fujioka S, Takatsuto S, Sakurai A, Feldmann KA. The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell. 1998;10(2):231–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Bancoş S, Nomura T, Sato T, Molnár G, Bishop GJ, Koncz C, et al. Regulation of transcript levels of the Arabidopsis cytochrome p450 genes involved in brassinosteroid biosynthesis. Plant Physiol. 2002;130(1):504–13.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Yu X, Li L, Zola J, Aluru M, Ye H, Foudree A, et al. A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 2011;65(4):634–46.

    Article  CAS  PubMed  Google Scholar 

  76. Oh E, Zhu J, Wang Z. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol. 2012;14(8):802–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Guo H, Ye H, Li L, Yin Y. A family of receptor-like kinases are regulated by BES1 and involved in plant growth in Arabidopsis thaliana. Plant Signal Behav. 2009;4(8):784–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Xing Y, Zhu Z, Wang F, Zhang X, Li B, Liu Z, et al. Role of calcium as a possible regulator of growth and nitrate nitrogen metabolism in apple dwarf rootstock seedlings. Sci Hortic. 2021;276(27):109740.

    Article  CAS  Google Scholar 

  79. Zhu X, Zhou P, Miao P, Wang H, Bai X, Chen Z, et al. Nitrogen use and management in orchards and vegetable fields in China: challenges and solutions. Front Agr Sci Eng. 2022;9(3):386–95.

    Google Scholar 

  80. Jia Z, Giehl RFH, Meyer RC, Altmann T, von Wirén N. Natural variation of BSK3 tunes brassinosteroid signaling to regulate root foraging under low nitrogen. Nat Commun. 2019;10(1):2378.

    Article  PubMed  PubMed Central  Google Scholar 

  81. Jia Z, Giehl RFH, von Wirén N. The root foraging response under low nitrogen depends on DWARF1-mediated brassinosteroid biosynthesis. Plant Physiol. 2020;183(3):998–1010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Chai S, Chen J, Yue X, Li C, Zhang Q, de Dios VR, et al. Interaction of BES1 and LBD37 transcription factors modulates brassinosteroid-regulated root forging response under low nitrogen in Arabidopsis. Front Plant Sci. 2022;13:998961.

    Article  PubMed  PubMed Central  Google Scholar 

  83. Liu Y, Wang H, Jiang Z, Wang W, Xu R, Wang Q, et al. Genomic basis of geographical adaptation to soil nitrogen in rice. Nature. 2021;590(7847):600–5.

    Article  CAS  PubMed  Google Scholar 

  84. Wani AS, Tahir I, Ahmad SS, Dar RA, Nisar S. Efficacy of 24-epibrassinolide in improving the nitrogen metabolism and antioxidant system in chickpea cultivars under cadmium and/or NaCl stress. Sci Hortic. 2017;225(18):48–55.

    Article  CAS  Google Scholar 

  85. Xing J, Wang Y, Yao Q, Zhang Y, Zhang M, Li Z. Brassinosteroids modulate nitrogen physiological response and promote nitrogen uptake in maize (Zea mays L). Crop J. 2022;10(1):166–76.

    Article  Google Scholar 

  86. Zhang S, Li J, Li M, Wang W, Wang D, Chang J, et al. Effects of exogenous 2,4-epibrassinolide on nitrogen metabolism and nicotine metabolism in flue-cured tobacco at mature stage. Tob Sci Tech. 2022;55(12):1–8.

    Google Scholar 

  87. Chen Y, Ren K, He X, Gong J, Hu X, Su J, et al. Dynamic changes in physiological and biochemical properties of flue-cured tobacco of different leaf ages during flue-curing and their effects on yield and quality. BMC Plant Biol. 2019;19(1):555.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We appreciate all the people who have collaborated on this project.

Funding

This work was supported by the Key R&D and Promotion Project of Henan Province (242102110288), the Scientific and Technological Project of Sanmenxia Tobacco Company (2024411200200016X), the Scientific and Technological Project of Luoyang Tobacco Company (2024410300270126), the Postdoctoral Fellowship Program (Grade C) of China Postdoctoral Science Foundation (GZC20230719). The funding bodies played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.

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  1. Haiyang Chen and Shuaitao Zhang contributed equally to this work.

Authors and Affiliations

  1. College of Tobacco Science, Henan Agricultural University, Zhengzhou, 450002, China

    Haiyang Chen, Shuaitao Zhang, Hongru Wei, Chaoyang Li, Zhaojun Wang & Xiaoquan Zhang

  2. Sanmenxia Branch of Henan Provincial Tobacco Corporation, Sanmenxia, 472000, China

    Jianbo Chang, Hongchen Li & Junjie Yang

  3. Luoyang Branch of Henan Provincial Tobacco Corporation, Luoyang, 471026, China

    Zhengxiong Song & Lihua Li

  4. College of Agronomy, State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou, 450046, China

    Xuelin Zhang

  5. Postdoctoral Station of Crop Science, Henan Agricultural University, Zhengzhou, 450046, China

    Haiyang Chen

  6. China Tobacco Henan Industrial Co., Ltd, Zhengzhou, 450002, China

    Jin Lun

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  1. Haiyang Chen
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Contributions

CHY and ZST performed the work and initiated the draft. CJB, WHR, LHC and LCY analyzed the data and prepared the figures and illustrations. YJJ, SZX, WZZ and LJ contributed the materials. CHY wrote the manuscript. ZXL and LLH help to design the experiment. CHY and ZXX obtained funding, designed experiments and revised the final version. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xuelin Zhang, Lihua Li or Xiaoquan Zhang.

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Chen, H., Zhang, S., Chang, J. et al. Foliar application of 24-epibrassinolide enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco by regulating cold stress tolerance. BMC Plant Biol 25, 77 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06080-1

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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