Synergistic effects of melatonin and chitosan in alleviating drought stress in saffron: (Crocus sativus) insights into plant growth and biochemical responses

Abstract

Saffron (Crocus sativus L.) is recognized as a strategic crop with high economic, nutritional, and cultural value in Iran; however, in recent years, its yield has significantly declined due to climate change, particularly drought stress. This study was conducted to evaluate the combined effects of drought stress at three field capacity (FC) levels (100%, 70%, and 40%), melatonin (100, 200, and 400 mg/L), and chitosan (100, 150, and 200 mg/L) on saffron performance over two years. The measured traits included leaf dry weight, leaf length, stigma dry weight, fresh flower weight, flower number, total corm weight, photosynthetic pigments, catalase activity, proline, electrolyte leakage (EL), and relative water content (RWC). Our results showed that the FC40 DS level had the most pronounced effects, significantly reducing RWC and pigment content, increasing EL, and ultimately leading to considerable reductions in leaf dry weight, leaf length, stigma dry weight, flower number, and total corm weight across both years. In contrast, the combined application of melatonin and chitosan significantly alleviated these adverse effects. The synergistic action of these two compounds helped maintain RWC, reduce EL, and enhance catalase activity, thereby mitigating reductions in both vegetative and reproductive performance particularly under FC70 and FC100. A strong positive correlation between stigma dry weight and flower number (R² = 0.79) indicated a close relationship between improved physiological processes and reproductive performance. Moreover, multivariate analysis using principal component analysis clearly distinguished the applied treatments and associated traits. “PC1 and PC2 explained 74.8% and 18.9% of variance respectively, clearly separating drought and biostimulant combinations based on physiological traits.” These findings confirm the synergistic application of melatonin and chitosan as an effective strategy to enhance saffron resilience under drought stress conditions.

Introduction

Saffron (Crocus sativus L.) ranks among the most valuable medicinal and spice plants globally, largely due to its bioactive constituents such as crocin, picrocrocin, and safranal, which play essential roles in the food, pharmaceutical, and cosmetic industries [17]. Predominantly cultivated in arid and semi-arid regions like Iran, this crop accounts for over 90% of the world's saffron production, serving as a critical income source for farmers in these areas [20]. From an economic standpoint, saffron’s high stigma value and its significance in sustaining rural livelihoods in dry regions necessitate innovative approaches to enhance production sustainability [20, 36]. Amidst dwindling water availability and rising global demand, the adoption of strategies centered on biological stimulants could be pivotal in improving both yield and quality. Nevertheless, drought stress (DS) remains a key environmental challenge, disrupting physiological functions such as photosynthesis, osmotic balance, and cellular membrane stability, thereby adversely affecting both vegetative and reproductive growth in saffron [9]. Given the ongoing climatic shifts and declining water resources, drought management in saffron cultivation has emerged as a critical focus for research and agricultural practice.

Drought is one of the foremost agricultural constraints in dry and semi-dry zones, reducing soil water potential and impairing vital plant processes including water uptake, nutrient transport, and photosynthesis [15]. In saffron, drought stress is particularly detrimental during sensitive growth phases like flowering and daughter corm formation. Water scarcity triggers stomatal closure and limits CO2 uptake, resulting in lowered photosynthetic rates and carbohydrate synthesis [32]. This negatively impacts vegetative growth traits such as leaf dry weight and length, as well as reproductive parameters including stigma dry weight, fresh flower weight, and flower number. Additionally, the reduction in daughter corm weight, which is crucial for subsequent production cycles, is a significant concern [3]. For instance, research has demonstrated that severe drought conditions (40% field capacity) can decrease saffron stigma yield by up to 60%, thereby directly affecting the crop’s economic value [3, 9]. Drought stress intensifies oxidative damage by promoting the accumulation of reactive oxygen species (ROS) such as superoxide radicals and hydrogen peroxide. These reactive molecules compromise cellular membranes, proteins, and DNA integrity, leading to increased electrolyte leakage (EL) and diminished membrane stability [6, 31]. In saffron, such damage correlates with a decline in relative water content (RWC) and elevated proline accumulation, which acts as an osmoprotectant, reflecting the plant’s effort to maintain osmotic balance [29]. However, under severe drought conditions, the plant’s antioxidant defenses particularly enzymes like catalase (CAT) and superoxide dismutase often prove insufficient, resulting in reduced productivity [32]. A recent study highlighted that drought stress limits carbohydrate synthesis in saffron by lowering chlorophyll content and inhibiting Rubisco enzyme activity, thereby decreasing stigma dry weight and flower number [17]. These detrimental effects underscore the urgent need for innovative strategies to alleviate drought impacts. In this context, emerging approaches involving growth regulators such as melatonin and biostimulants like chitosan have gained attention for their potential to enhance drought tolerance and improve crop performance [29, 38]. Melatonin, a multifunctional molecule known for its antioxidant properties and regulatory role in plant growth, has been recognized over the past decade as a crucial agent in boosting plant resilience against environmental stresses [6, 8]. By scavenging ROS, modulating the expression of photosynthesis-related genes, and stimulating the synthesis of osmolytes like proline, melatonin mitigates the adverse effects of DS [4, 21]. In saffron specifically, melatonin application enhances antioxidant enzyme activities, including catalase and peroxidase, thereby reducing oxidative injury and preserving membrane integrity [29]. For instance, foliar treatment with 100 μM melatonin under DS conditions has been reported to sustain chlorophyll levels and prevent reductions in leaf and stigma dry weights [29]. Moreover, melatonin influences hormonal signaling pathways such as abscisic acid and jasmonic acid, which strengthen the plant’s defense mechanisms [39]. These regulatory effects help maintain relative water content and improve water use efficiency, supporting both vegetative and reproductive growth during stress [21]. Additionally, melatonin upregulates genes involved in proline biosynthesis, contributing to osmotic homeostasis and alleviating drought-induced osmotic stress at the cellular level [1, 7]. Such properties position melatonin as a promising tool for managing DS in saffron, particularly in water-limited environments.

Chitosan, a natural polysaccharide derived from chitin, has attracted considerable interest in agriculture due to its biocompatibility, biodegradability, and antimicrobial properties [38]. This compound enhances plant resilience to environmental stresses by promoting nutrient uptake, reinforcing cell walls, and stimulating the synthesis of secondary metabolites [35]. In saffron, chitosan application has been shown to boost antioxidant enzyme activities, including catalase and superoxide dismutase, thereby alleviating oxidative damage caused by ROS and improving membrane stability [9]. Recent findings indicate that foliar spraying of chitosan at 150 mg/L under DS reduces electrolyte leakage by up to 25% and prevents declines in stigma dry weight [38]. Furthermore, chitosan activates defense signaling pathways such as the Jasmonic acid route, enhancing the biosynthesis of bioactive compounds like crocin in saffron, which contributes to stigma quality improvement [28]. By increasing membrane permeability and stimulating root growth, chitosan facilitates greater water and nutrient absorption particularly nitrogen and potassium helping to maintain relative water content during stress [35]. For example, a 2023 study demonstrated that chitosan improves nitrogen metabolism in saffron by elevating nitrate reductase activity, resulting in increased daughter corm weight [3, 9]. These attributes make chitosan a valuable complementary strategy for managing DS in saffron cultivation. Despite advancements in understanding DS effects on saffron and the individual roles of melatonin and chitosan in mitigating these impacts, significant knowledge gaps remain. Most previous research has focused on the separate effects of melatonin or chitosan [29, 38]. However, information on their combined, potentially synergistic effects under drought conditions in saffron is scarce. Moreover, the majority of studies have been conducted under controlled greenhouse environments, with limited data available regarding the application of these compounds under field conditions over extended periods, such as multiple growing seasons [3]. Additionally, the integrated influence of these treatments on key physiological parameters such as chlorophyll content, catalase activity, proline accumulation, electrolyte leakage, and relative water content has not been extensively explored in saffron [9]. Addressing these gaps highlights the need for comprehensive field-based investigations to evaluate the combined effects of DS, melatonin, and chitosan treatments, with a focus on elucidating underlying physiological mechanisms.

Material and methods

This experiment was conducted over two consecutive years (2021 and 2022) in Bahabad County, Yazd Province, Iran, on a plot of approximately 1,000 square meters. The experimental design followed a randomized complete block design (RCBD) in a split-factorial arrangement with three replications. The main plots consisted of three irrigation levels (40%, 70%, and 100% of field capacity), while the subplots included various combinations of chitosan at concentrations of 100, 150, and 200 mg/L, and melatonin at three levels: 100, 200, and 400 µM. In other words, the subplot treatments involved simultaneous combinations of the two substances at different levels, and no individual treatments of chitosan or melatonin alone were applied. 100 mg/L melatonin, 100 mg/L chitosan and 100% FC was considered as control treatment. Prior to planting, the field was thoroughly plowed and amended with 40 tons per hectare of well-rotted cattle manure. Certified saffron corms, provided by the Medicinal Plants Research Institute of Ardakan University, were used for planting. After removing adhered soil, the average weight of the corms was approximately 8 g. The planting was carried out with 20 cm between rows and 10 cm between corms within each row, at a planting depth of 20 cm. Given the plot dimensions (1 × 3 m), four rows were established in each plot. The first irrigation was applied three weeks before the air temperature dropped to 12 °C. Saffron corms were planted in the first week of September. Melatonin and chitosan were applied via foliar spray in mid-October, approximately three weeks after emergence, Morphological data were collected during peak flowering in early November, and physiological traits (e.g., RWC, EL, CAT) were assessed during mid to late flowering stages. Morphological traits (LDW, LL, FN, SDW) were measured at flowering peak, Physiological traits (RWC, EL, CAT, PRO, pigments) were assed at mid and late flowering stages.

Soil physicochemical characteristics and meteorological information

Planting was carried out in soil with a loam texture. The soil physical and chemical characteristics are given in Table 1. Using hand hoes, weed control was performed manually. During the trial period, the air temperature and precipitation data (Fig. 1) were recorded using a weather data logger.

Table 1.

The soil physical and chemical characteristics of the experimental

Depth	pH	ECe	Organic carbon (OC)	Total N	Available P	Available K	Clay	Sand	Silt	Texture
		mS.cm−1	%	%	mg.kg−1	mg.kg−1	%	%	%
0–30 cm	7.2	4.3	0.41	0.02	11.9	236	21	41	38	Loam

Fig. 1.

The thermo-pluviometric trend diagram at the research field (Bahabad, Yazd province, Iran) during 2021–2022

Measured traits

Leaf dry weight

To measure the leaf dry weight of saffron plants, fresh leaves were carefully detached and dried in an oven at 60–70 °C for 48 h until complete moisture removal. The dried samples were then weighed using a precision balance with an accuracy of 0.001 g.

Leaf length

To determine leaf length, healthy and fully developed leaves were detached from the plants and measured from the base (point of attachment to the corm) to the tip using a digital caliper with 0.1 mm precision. This measurement was performed on randomly selected samples, and the average leaf length was calculated.

Stigma dry weight

For stigma dry weight, fresh stigmas were carefully separated from flowers after harvest and dried in an oven at 40–50 °C for 24–48 h to ensure complete dehydration. The dried stigmas were then weighed using a precision balance (accuracy of 0.001 g).

Fresh flower weight

Fresh, healthy saffron flowers were collected immediately after harvest and weighed using a digital scale with an accuracy of 0.01 g to determine fresh flower weight.

Flower number

To count flower number, fully opened flowers per unit area (typically per square meter) were recorded during the peak flowering period. Daily field inspections were carried out, and the number of produced flowers was recorded over a defined period.

Total corm weight

To assess total corm weight, healthy and intact corms were harvested, cleaned to remove soil and roots, and weighed using a precision balance with 0.01 g accuracy.

Relative water content

Relative water content was determined based on the method of Smart and Bingham [37]. Leaf discs (8 mm) were weighed fresh (WF), then soaked in distilled water for 2–3 h to obtain turgid weight (WT), and finally dried at 80 °C for 24 h to record dry weight (WD). RWC was calculated using: RWC = [(WF – WD)/(WT – WD)] × 100, indicating leaf hydration status.

Electrolyte leakage

To estimate electrolyte leakage (EL), the procedure outlined by Lutts et al. [27] was applied. In this method, five 8-mm leaf discs from each treatment were placed in 15 mL of distilled water and gently agitated at room temperature for 24 h. The initial electrical conductivity (EC₁) of the solution was then measured. Following this, samples were sterilized in an autoclave at 121 °C for 30 min. Once cooled, the final conductivity (EC₂) was determined. The extent of ion leakage was calculated using the formula: EL = (EC₁/EC₂) × 100, representing the percentage of ions released from damaged cell membranes.

Photosynthetic pigments

Photosynthetic pigments were quantified following Lichtenthaler and Buschmann [25]. Fresh leaf tissue (0.1 g) was extracted using 80% acetone, and after centrifugation at 6000 rpm, absorbance was recorded at 663 nm (chlorophyll a), 645 nm (chlorophyll b), and 470 nm (total carotenoids) using a microplate reader (EON, Biotech, USA). Concentrations of chlorophyll a, chlorophyll b, and total carotenoids (mg/g fresh weight) were calculated using established equations incorporating absorbance values, extraction volume, and sample weight.

Proline quantification

To assess proline levels, the protocol described by Bates et al. [11] was adapted. Initially, 0.05 g of finely ground and dried plant tissue was mixed with 5 mL of a 3% sulfosalicylic acid solution and thoroughly homogenized using a porcelain mortar. The resulting homogenate was left to stand for 48 h before filtration. Then, a 1 mL aliquot of the filtrate was combined with 1 mL of ninhydrin reagent and 1 mL of glacial acetic acid. The mixture was incubated in a boiling water bath for one hour. After allowing the samples to cool on ice, 2 mL of toluene was added, and the solution was vigorously shaken for 5 to 20 s. The upper toluene layer was separated and its absorbance was read at 520 nm to determine proline concentration.

Catalase activity

CAT activity was evaluated by tracking the decline in H₂O₂ absorbance at 240 nm over a 30-s period [14]. The reaction mixture (3 mL total volume) included 50 mM phosphate buffer (pH 7.0), 15 mM hydrogen peroxide, and 100 μL of enzyme extract. The reaction began upon addition of H₂O₂, and absorbance was recorded immediately. Enzyme activity was expressed as the amount of catalase that breaks down 1 μmol of H₂O₂ per minute, using an extinction coefficient of 40 mM⁻¹ cm⁻¹ for calculation.

Statistical analysis

The experiment was designed to evaluate three factors: drought levels, chitosan levels, and melatonin levels, each with three treatment levels. The analysis was performed using a factorial model based on a split-factorial arrangement within a randomized complete block design (RCBD) with three replications. Data analysis was conducted in R software. A variance analysis (ANOVA) using the F-test was conducted to evaluate the effects of treatments at each sampling interval, with means compared using Fisher's Least Significant Difference (LSD) test at the 5% significance level. Principal component analysis (PCA) was applied to interpret the physiological response patterns. Additionally, in this study, the relationships among the evaluated traits were analyzed using the Pearson correlation coefficient and Linear regression models were employed to identify relationships between physiological parameters with the ggplot2 package in R. Prior to combined analysis, Levene’s test for homogeneity of variances was conducted. As variances were significantly different between years for some traits, the data were analyzed separately for each year. Only PCA and correlation analyses were performed on the pooled dataset for comparative purposes.

Results

Leaf Dry Weight (LDW)

In the first year, leaf dry weight (LDW) was evaluated under different field capacity (FC) levels (FC: 100%, 70%, 40%), melatonin concentrations (M: 100, 200, 400 mg/L), and chitosan doses (CH: 100, 150, 200 mg/L). LDW ranged from 0.313 to 3.300 g across treatments. The highest LDW (3.300 g) was observed under FC40 + M100 + CH100 (Fig. 2A). This was followed by FC70 + M400 + CH100 (2.933 g), and two treatments FC100 + M100 + CH200 and FC70 + M200 + CH200 both with 2.567 g. Other effective treatments included FC70 + M100 + CH150 (2.500 g) and FC70 + M400 + CH200 (1.967 g), indicating positive responses under FC70. In contrast, the lowest LDW was recorded in FC40 + M400 + CH200 (0.313 g), followed by FC40 + M100 + CH200 (0.467 g), FC40 + M200 + CH200 (0.627 g), FC40 + M400 + CH100 (0.650 g), and FC40 + M400 + CH150 (0.703 g), all under FC40 (Fig. 2A).

Fig. 2.

Effects of irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L) on (A) leaf dry weight (LDW), B leaf length (LL), C stigma dry weight (SDW), D fresh flower weight (FFW) and (E) flower number (FN) in saffron at first years. The bars represent the ± SD and different letters represent significant differences (P < 0.05) within the same group

Leaf Length (LL)

Leaf length LL values ranged from 13.49 to 23.84 cm across treatments. Statistical analysis revealed that the differences among treatments were significant (P < 0.001) (Fig. 1B). The highest LL (23.84 cm) was recorded under FC100 + M100 + CH100, followed by FC100 + M400 + CH150 (23.18 cm) and FC100 + M200 + CH200 (23.15 cm), ranked second and third, respectively. Other treatments with relatively high LL included FC100 + M400 + CH200 (22.94 cm), FC40 + M100 + CH150 (22.68 cm), and FC100 + M200 + CH100 (22.12 cm). Conversely, the lowest LL was observed in FC40 + M400 + CH150 (13.49 cm), followed by FC40 + M200 + CH100 (15.28 cm) and FC40 + M200 + CH150 (15.98 cm). Additionally, treatments such as FC40 + M400 + CH200 (16.14 cm), FC70 + M100 + CH200 (16.46 cm), and FC100 + M400 + CH100 (16.86 cm) were also among those with lower LL values (Fig. 2B).

Stigma Dry Weight (SDW)

In the first year, the effect of various treatments on stigma dry weight (SDW) was evaluated, with values ranging from 0.230 to 0.427 g (Fig. 1C). The highest SDW (0.427 g) was recorded under the treatment FC100 + M100 + CH100. In contrast, the lowest SDW (0.230 g) was observed under FC40 + M200 + CH150. Overall, reducing FC from 100 to 40% noticeably decreased SDW, especially when higher levels of melatonin and chitosan were applied. However, treatments under FC70 and FC100 generally showed relatively better performance. It is noteworthy that the was 0.387 g in the FC70 + M200 + CH200 treatment and 0.389 g in the FC70 + M400 + CH200 treatment.

Fresh Flower Weight (FFW)

The fresh flower weight (FFW) among treatments ranged from 14.87 to 28.47 g (Fig. 2D) Notably, under FC70 combined with high concentrations of melatonin and chitosan, a substantial increase in FFW was observed. In contrast, FC40 along with high doses of melatonin and chitosan in certain treatments resulted in a significant reduction in this trait. In the first year of the experiment, the highest FFW was recorded under the FC70 + M400 + CH100 treatment, reaching 28.47 g. This was followed by FC70 + M200 + CH200 with 25.87 g and FC100 + M200 + CH100 with 26.20 g, ranking second and third, respectively. Other high-performing treatments included FC100 + M100 + CH100 (25.07 g), FC40 + M100 + CH100 (24.90 g), and FC70 + M400 + CH200 (24.93 g). Conversely, the lowest FFW was observed under the FC40 + M400 + CH100 treatment with 14.87 g. The FC40 + M100 + CH200 treatment recorded the second lowest value at 16.03 g. Additional treatments with low performance in this trait included FC40 + M200 + CH200 (16.33 g), FC100 + M400 + CH100 (16.57 g), and FC70 + M200 + CH150 (17.43 g) (Fig. 2D).

Flowers Number (FN)

The number of flowers per plant varied widely among treatments, ranging from 24.67 to 84.33, reflecting the substantial impact of combined irrigation, melatonin, and chitosan treatments on flower production (Fig. 2E). Notably, FC70 combined with melatonin and chitosan significantly enhanced flowering. The FC70 + M400 + CH100 treatment recorded the highest FN (84.33), clearly demonstrating the beneficial effect of FC70 in combination with low doses of melatonin and chitosan on FN. This was followed by FC100 + M100 + CH100 (75.67 flowers) and FC100 + M400 + CH150 (73), which ranked second and third, respectively. Other treatments with high FN included FC100 + M200 + CH200 (70.33), FC100 + M400 + CH200 (68), and FC70 + M200 + CH200 (68.67), highlighting their positive influence on FN. Conversely, the lowest FN was observed under FC40 + M400 + CH150 with 24.67 FN. Other low-performing treatments included FC40 + M400 + CH100 (25), FC40 + M200 + CH100 (26.67), FC40 + M100 + CH200 (28), and FC40 + M400 + CH200 (29.67).

Leaf dry weight

Figure 2 illustrates the variation in leaf dry weight (LDW) during the second year, ranging from 1.30 to 8.57 g (Fig. 3A. (Overall, the combination of FC100 with high levels of melatonin and chitosan had the most pronounced positive effect on LDW. In contrast, FC40, particularly when combined with lower levels of these substances, led to a significant reduction in growth. The highest LDW was recorded under the FC100 + M400 + CH200 treatment, with a value of 8.57 g. It was followed by FC70 + M100 + CH100 (7.13 g) and FC100 + M400 + CH100 (7.07 g), ranking second and third, respectively. Additionally, treatments such as FC100 + M200 + CH150 (6.83 g) and FC100 + M400 + CH150 (6.87 g) also recorded high LDW. Conversely, the lowest LDW was observed in the FC40 + M100 + CH200 treatment, which yielded just 1.30 g. Other low-performing treatments included FC40 + M100 + CH150 (1.47 g), FC40 + M400 + CH150 (1.83 g), and FC40 + M200 + CH200 (2.33 g) (Fig. 3A).

Fig. 3.

Effects of irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L) on (A) leaf dry weight (LDW), B total corm weight (TCW), C leaf length (LL), D fresh flower weight (FFW), E flower number (FN) and (F) corm weight (CW) in saffron at second years. The bars represent the ± SD and different letters represent significant differences (P < 0.05) within the same group

Total Corm Weight (TCW)

In the second year of the experiment, the evaluation of different combinations of FC, melatonin and chitosan revealed significant effects on total corm weight (TCW) (Fig. 3B). The TCW values ranged from 19.20 to 67.83 g across treatments. The highest of TCW was obtained under the FC100 + M100 + CH100 treatment, with an average of 67.83 g. Notably, treatments such as FC70 + M100 + CH100 (53.73 g) and FC100 + M400 + CH150 (58.00 g). Conversely, the lowest corm weights were recorded for FC70 + M200 + CH150 (19.77 g) and FC40 + M100 + CH200 (19.20 g). Interestingly, some treatments like FC100 + M400 + CH100 (27.27 g) and FC70 + M400 + CH100 (24.40 g) (Fig. 3B).

Leaf Length

The analysis of leaf length (LL) data in the second year revealed that the range of variation spanned from 17.73 cm to 37.05 cm. The highest average (LL) was recorded for the FC100 + M100 + CH100 treatment, reaching 37.05 cm (Fig. 3C). Other treatments with notable results included FC70 + M400 + CH100 (35.23 cm), FC100 + M200 + CH100 (34.81 cm), and FC70 + M100 + CH150 (34.5 cm), all of which contributed significantly to increased LL. In contrast, the shortest LL was observed in the FC40 + M400 + CH150 treatment (17.73 cm). Similarly, the treatments FC40 + M200 + CH150 (19.67 cm) and FC40 + M400 + CH100 (20.27 cm) also showed poor performance in terms of LL.

Stigma Dry Weight (SDW)

The range of variation in Stigma Dry Weight (SDW) extended from 0.26 to 0.51 g ((Fig. 3C). The highest SDW was recorded in the FC100 + M100 + CH100 treatment, with an average of 0.51 g. Similarly, the treatments FC70 + M100 + CH150 (0.49 g) and FC100 + M200 + CH100 (0.49 g). On the other hand, the lowest SDW was observed in the FC40 + M200 + CH150 treatment, with an average of 0.26 g. Other treatments such as FC100 + M400 + CH100 and FC40 + M100 + CH200 also showed weak performance, each recording an average of 0.29 g.

Fresh flower weight

The results from the evaluation of fresh flower weight (FFW) in the second year revealed that this trait was significantly influenced by different irrigation, melatonin, and chitosan treatments. The average FFW across treatments ranged from 18.83 to 36 g (Fig. 2D). The highest FFW was recorded in the FC100 + M200 + CH200 treatment, with a mean of 36 g. Additionally, the treatments FC70 + M100 + CH100 and FC100 + M100 + CH150 showed good performance, with average values of 35.8 g and 34.07 g, respectively. On the other hand, the lowest FFW was observed in the FC40 + M200 + CH200 treatment, with an average of 18.83 g.

Flower number

The evaluation of flower number (FN) in the second year demonstrated that different treatments had varying effects on flower production. Among all treatments, FC100 + M100 + CH100 recorded the highest average FN with 92 per plant (Fig. 3E). Other FC100 treatments also showed favorable results, with FC100 + M200 + CH100 averaging 88.67 flowers and FC100 + M400 + CH100 producing 55.67 flowers. Under FC70 combined with varying levels of melatonin and chitosan, the treatment FC70 + M400 + CH100 achieved nearly comparable performance with 91 flowers, while FC70 + M100 + CH200 also performed relatively well with 64.33 flowers. In contrast, treatments under FC40 combined with high melatonin and chitosan levels resulted in lower FN. Specifically, FC40 + M100 + CH200 and FC40 + M200 + CH150 recorded the lowest FN, with 36 and 30.67 flowers, respectively.

Average corm weight (CW)

The evaluation of corm weight (CW) in the second year revealed varied results across treatments. The treatment FC70 + M100 + CH150 recorded the CW at 9.45 g (Fig. 3F). Specifically, FC100 + M100 + CH100 and FC100 + M200 + CH150 achieved notable weights of 7.77 g and 7.11 g, respectively, while FC100 + M100 + CH200 exhibited the lowest value at 4.61 g. Under treatments of FC70 + M100 + CH150 again ranked highest with 9.45 g, whereas other treatments such as FC70 + M100 + CH100 (6.67 g) and FC70 + M200 + CH100 (6.22 g) attained fairly good bulb weights. In the low FC40, the highest CW was recorded by FC40 + M100 + CH150 with 8.08 g, while FC40 + M100 + CH200 showed the lowest weight at 4.72 g.

Chlorophyll a concentration

The assessment of chlorophyll a (Chl a) concentration revealed varied effects of different treatments on its levels. The treatment FC70 + M200 + CH100 recorded the highest Chl a concentration at 1.26. Treatments FC70 + M100 + CH150 (0.81) and FC70 + M100 + CH200 (0.75) also showed good performance in increasing chlorophyll a concentration. In contrast, the lowest concentrations were observed in FC100 + M100 + CH150 (0.29) and FC40 + M200 + CH150 (0.26). Additionally, treatments FC40 + M100 + CH200 (0.98) and FC40 + M100 + CH150 (0.69) fell within the higher concentration category (Fig. 4A).

Fig. 4.

Effects of irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L) on (A) chlorophyll a (Chl a), B chlorophyll b (Chl b), C total chlorophyll a (Chl a), D catalase (CAT), E proline (PRO) (F) electrolyte leakage (EL), G relative water content (RWC) and stigma dry weight (SDW) in saffron at second years. The bars represent the ± SD and different letters represent significant differences (P < 0.01) within the same group

Chlorophyll b Concentration

In this study, the treatment FC70 + M200 + CH100 exhibited the highest chlorophyll b (Chl b) concentration at 0.66. Additionally, treatments FC70 + M100 + CH150 (0.61) and FC70 + M400 + CH200 (0.65) also showed notable performance in increasing Chl b concentration. On the other hand, the treatment FC70 + M400 + CH150 recorded the lowest Chl b concentration at 0.18, reflecting the negative impact of certain combinations in this treatment. Moreover, FC100 + M100 + CH150 with 0.20 was among the treatments with lower Chl b concentrations (Fig. 4B).

Total chlorophyll content

In this study, the treatment FC70 + M200 + CH100 showed the highest total chlorophyll (TChl) content at 1.92. Following this, treatments FC70 + M200 + CH150 (1.63) and FC70 + M100 + CH150 (1.42) exhibited acceptable levels of TChl. Conversely, the treatment FC40 + M400 + CH200 recorded the lowest TChl content at 0.41 (Fig. 4C).

Catalase (CAT) Enzyme Activity

In this study, the highest catalase (CAT) enzyme activity was observed in the treatment FC70 + M400 + CH200, reaching a value of 34.5. The next highest activity was recorded in the treatment FC40 + M400 + CH100 with 32.9, indicating a significant increase in CAT activity in the plant under this combination as well. Treatments FC70 + M200 + CH100 with 31.1 and FC100 + M100 + CH150 with 31.3 also significantly enhanced CAT enzyme activity compared to other treatments (Fig. 4D). Conversely, the treatment FC40 + M100 + CH200 showed the lowest CAT activity at 21.3, likely due to the negative impact of reduced irrigation (FC40) on the efficiency of the plant’s defense systems. Overall, the results indicate that the combinations FC70 + M400 + CH200 and FC40 + M400 + CH100 had the greatest effect in enhancing CAT enzyme activity in the plants.

Proline content

In this study, the treatment FC40 + M400 + CH200 showed the highest proline (PRO) accumulation with a value of 3.33. Following that, treatments FC70 + M200 + CH200 and FC40 + M400 + CH150 produced notable PRO levels of 3.17 and 3.03, respectively. Among other treatments, FC100 + M400 + CH200 with 2.7 and FC70 + M200 + CH100 with 3.03 also showed significant success in PRO production. Conversely, FC100 + M100 + CH100 had the lowest PRO content at 1.46 (Fig. 4E).

Electrolyte Leakage (EL)

In the assessment of electrolyte leakage (EL), which indicates cellular damage caused by environmental stresses, the treatment FC40 + M200 + CH150 showed the highest level of electrolyte leakage at 0.93. Following that, treatments FC40 + M200 + CH100 with 0.89 and FC40 + M100 + CH200 with 0.79 also exhibited high EL. On the other hand, treatments FC70 + M100 + CH100 with 0.84 and FC100 + M100 + CH100 with 0.41 showed relatively high levels of EL. Finally, treatments FC40 + M100 + CH150 with 0.41 and FC100 + M200 + CH150 with 0.46 (Fig. 4F).

Relative Water Content (RWC)

In the assessment of relative water content (RWC), which is an indicator of plant water status and its ability to retain moisture under various stress conditions, the treatment FC100 + M400 + CH100 showed the highest RWC at 93.3%. Comparatively, the treatment FC70 + M100 + CH150 with 94.3% was also identified as one of the treatments with high performance in moisture retention and optimal irrigation conditions. This treatment, along with FC100 + M100 + CH200, which achieved 92.83% RWC. Meanwhile, treatments such as FC40 + M400 + CH150 (with 84.17%) and FC70 + M400 + CH150 (with 83.57%) showed the lowest relative water content values (Fig. 4G).

Stigma dry weight (SDW)

The results from the second year regarding the stigma dry weight (SDW) of saffron revealed that the combined treatments of soil moisture levels, melatonin, and chitosan exerted varying effects on the plant's reproductive performance (P < 0.001). The highest SDW value (0.507 g) was recorded in the treatment FC100 + M100 + CH100. The highest SDW value (0.507 g) was recorded in the treatment FC100 + M100 + CH100. Under the same moisture level (FC100), other treatments also demonstrated acceptable performance. Under FC70, treatments FC70 + M100 + CH150 (0.490 g) and FC70 + M400 + CH100 (0.467 g) exhibited the highest SDW values. In contrast, under FC40, a general decline in SDW was observed. The highest SDW at this stress level was found in the treatment FC40 + M100 + CH100 (0.420 g), while the lowest value (0.260 g) was recorded in FC40 + M200 + CH150 (Fig. 4H).

Principal Component Analysis (PCA) Results

The PCA for the first year revealed that the first two principal components, PC1 and PC2, explained 74.8% and 18.9% of the total variance, respectively, highlighting their strong capability in data differentiation. Variables such as LDW and SDW were positioned negatively on both PC1 and PC2. Flower number (FN) and LL appeared positively on PC2 and near zero on PC1, reflecting their association with vegetative growth. Observations like FC100 + M400 + CH150 and FC70 + M200 + CH150 were scattered in the positive quadrant of PC1. Fresh flower weight (FFW) clustered near the origin, indicating a moderate influence on data separation. These patterns suggest that PC1 primarily relates to performance traits, while PC2 is associated with growth characteristics (Fig. 5A).

Fig. 5.

Principal component analysis (PCA) plot illustrating the morphological, physiological and biochemical responses of saffron at first years (A) and second years (B) under irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L). The plots feature PC1 and PC2 explained of the variance. Circles represent sample scores and arrows show gradients resulting

For the second year, PCA showed that PC1 and PC2 explained 36.6% and 18% of the total variance, respectively, demonstrating variable and observation dispersion across these components. Leaf dry weight (LDW), TCW, and FN were located negatively on PC1 and close to zero on PC2. Conversely, Chl a, Chl b, and CAT scattered positively across both PC1 and PC2, reflecting the influence of varying environmental conditions. Observations such as FC70 + M200 + CH150 and FC40 + M300 + CH150 clustered in the positive quadrant of both PCs. Proline (PRO) and SDW appeared near the positive side of PC2 and close to the PC1 axis. Leaf length (LL) and FFW were distributed near the origin, indicating a lesser role in data separation (Fig. 5B).

Correlation analysis

In this study, we examined the correlations between the measured traits in the first and second years of saffron cultivation. Our results showed that in the first year, there were significant positive correlations among all measured traits (Fig. 6-A). The highest correlation observed in the first year was between FFW and SDW (R² = 0.79) (Fig. 5-A). In contrast, in the second year, significant correlations were observed between FN and FFW (R² = 0.60), SDW (R² = 0.68), and LL (R² = 0.78) (Fig. 5-B). Additionally, significant positive correlations existed between LL and FFW (R² = 0.53) and between LL and SDW (R² = 0.55). Moreover, significant negative correlations were found between EL and LL (R² = −0.53), FFW (R² = −0.50), and FN (R² = −0.58). We also observed significant positive correlations between TChl and Chl a (R² = 0.65), as well as TChl and Chl b (R² = 0.54) (Fig. 6-B).

Fig. 6.

Correlation matrix of phenotypic traits in saffron under difference treatments during the first (A) and second (B) years. The color gradient, ranging from green to purple, illustrates the strength and direction of the correlations green indicating strong positive and purple indicating strong negative relationships. Numerical values within the matrix denote Pearson correlation coefficients calculated between traits. Asterisks indicate statistical significance levels of the correlations (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)

Relationship between traits

To explore the relationships between traits, a linear regression model was applied. Our results in the first year indicated a significant positive relationship between LDW and SDW (R² = 0.50). A positive relationship was also observed between LL and SDW (R² = 0.44). Furthermore, a significant positive correlation was found between FFW and SDW (R² = 0.67) (Fig. 7-C). Additionally, a positive correlation existed between FN and SDW (R² = 0.38) (Fig. 7A-D, respectively).

Fig. 7.

Relationships between stigma dry weight (SDW) whit (A) leaf dry weight (LDW), B leaf length (LL), C fresh flower weight (FFW) and (D) flower number (FN) after interaction irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L) at first years. The coefficients of determination (R.²) of the relationships are given (P < 0.01)

In the second year, positive correlations were observed between LL and SDW (R² = 0.51), FFW and SDW (R² = 0.20), and FN and SDW (R² = 0.60) (Fig. 7A-C, respectively). Moreover, a positive correlation was detected between TCW and SDW (R² = 0.17). Conversely, negative correlations were found between PRO and SDW (R² = 0.11) and between EL and SDW (R² = 0.39) (Fig. 8D-F, respectively).

Fig. 8.

Relationships between stigma dry weight (SDW) whit (A) leaf length (LL), B fresh flower weight (FFW), C flower number (FN), D total corm weight (TCW), E proline (PRO) and (F) electrolyte leakage (EL) after interaction irrigation levels 100% FC, 70% FC and 40% FC) melatonin (M-100, 200, and 400 mg/L), and chitosan (CH-100, 150, and 200 mg/L) at second years. The coefficients of determination (R.²) of the relationships are given (P < 0.01)

Discussion

Saffron as one of the most valuable medicinal and spice plants, is highly susceptible to drought stress (DS) due to its cultivation in arid and semi-arid regions [17]. Generally, DS limits the vegetative and reproductive growth of saffron by disrupting physiological and biochemical processes, including photosynthesis, osmotic balance, and membrane stability [5]. This study investigated the effects of DS at three field capacity levels (FC: 100%, 70%, 40%), melatonin (M-100, 200, 400 mg L⁻¹), and chitosan (CH-100, 150, 200 mg L⁻¹) over two years, demonstrating that these treatments significantly influenced vegetative traits, reproductive traits, corm weight, and physiological indices. Melatonin and chitosan acted through complementary mechanisms to mitigate the adverse effects of DS and enhance plant performance [38].

Drought stress impairs water and nutrient uptake by reducing soil water potential, leading to disturbances in key physiological processes [15]. In saffron, this effect manifested as a reduction in LDW across both years. Drought stress limits photosynthesis by decreasing turgor pressure and inducing stomatal closure, which restricts CO₂ uptake [23]. This phenomenon was evident in the FC40 treatments, which showed decreased total chlorophyll concentration in the second year and reduced LDW, attributed to chlorophyll degradation and diminished Rubisco activity, thereby limiting carbohydrate synthesis. The carbohydrate shortage not only curtailed vegetative growth (LDW and LL) but also impaired the translocation of nutrients to reproductive organs (SDW, FFW, FN) and storage tissues (TCW), consistent with Ahmadian et al. [3], who reported that DS limits saffron stigma yield via photosynthetic inhibition.

Furthermore, DS reduces cellular hydration by lowering water potential, thereby decreasing (RWC. Our findings revealed that the FC40 treatments combined with low concentrations of chitosan and melatonin resulted in a reduced RWC in the second year. This reduction leads to a loss of turgor pressure and inhibits cell division and elongation, causing decreased LL and FFW [32]. Additionally, drought-induced oxidative stress elevates ROS such as superoxide and hydrogen peroxide, damaging cellular membranes. Consequently, EL significantly increased in FC40 treatments with low chitosan and melatonin concentrations during the second year, indicating compromised membrane integrity, which restricted reproductive performance (SDW and FN) [10]. Drought stress also exacerbates oxidative stress, as evidenced by increased proline accumulation and CAT activity in FC70 and FC40 treatments during the second year. Proline, as an osmoprotectant, provides antioxidant properties by scavenging free radicals. However, insufficient antioxidant defense under FC40 can result in protein and lipid damage [30]. Proline can maintain turgor pressure, thereby facilitating adequate conductance for respiratory gases to mitigate water deficiency. This mechanism helps sustain high levels of CO₂ in the intercellular spaces of mesophyll cells, preventing or reducing the inhibition of photosynthesis in photosynthetic organs [34]. The results of the present study demonstrate that the applied treatments led to an increase in proline concentration under drought stress. These findings suggest that proline stabilizes proteins and cellular membranes, preventing their denaturation under water-deficit conditions. This property contributes to maintaining the structural integrity of the photosynthetic apparatus and other cellular structures, which is essential for sustaining cellular hydration and function [18, 24]. Additionally, it has been reported that osmotic adjustment of inorganic ions in plants is highly dependent on ion pumps, such as those for Na⁺, K⁺, and H⁺, which regulate the concentration of these ions inside and outside the cell, thereby altering cellular osmotic potential and affecting cell shape and function. The accumulation of K⁺ and free proline plays a critical role in plant adaptation to drought stress, while Na⁺ accumulation is one of the most effective strategies for drought tolerance [16, 19, 34]. These physiological disruptions led to reductions in corm weight and reproductive yield, as the plant reallocates energy from reproductive growth to defense responses [5].

Melatonin, as a multifunctional molecule with antioxidant properties and growth-regulating functions, modulates the adverse effects of DS in saffron through multiple mechanisms [8]. The results of this study demonstrated that melatonin treatments at M100 and M200 dose, particularly under FC100 and FC70 conditions, significantly enhanced both vegetative and reproductive performance. For instance, the FC100 + M100 + CH100 treatment exhibited the highest LL, SDW, and FN, underscoring melatonin’s role in strengthening physiological processes.

Melatonin aids plants in coping with drought stress through multiple physiological, biochemical, molecular, and metabolic mechanisms. Physiologically, melatonin regulates stomatal behavior, thereby maintaining the balance between transpiration and photosynthesis and reducing water loss. For instance, Ahmad et al. [2] demonstrated that melatonin improved water use efficiency in maize by reducing stomatal opening. Moreover, melatonin preserves photosynthetic efficiency by protecting chloroplasts and enhancing chlorophyll content. Imran et al. [21] reported that the application of 100 µM melatonin in soybean increased chlorophyll content and photosynthetic rate under drought conditions. In addition, melatonin promotes root growth, which enhances water uptake under water-deficit conditions. Altaf et al. [4] showed that increased root length and volume in tomato contributed to improved drought tolerance. Due to its antioxidant capacity, melatonin enhances the antioxidant defense system and reduces oxidative stress. Our findings showed that the FC70 + M400 + CH200 treatment resulted in the highest CAT activity and that FC100 + M100 + CH100 significantly decreased EL, indicating melatonin’s protective role in maintaining membrane integrity. From a biochemical perspective, melatonin functions as a potent antioxidant by directly scavenging reactive oxygen species (ROS) and enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD), CAT, and peroxidase (POD), thereby mitigating oxidative damage to membranes, proteins, and DNA. Jafari and Shahsavar et al. [22] reported that melatonin preserved membrane stability in lemon. Furthermore, melatonin contributes to osmotic adjustment by promoting the synthesis of compatible solutes such as proline, soluble sugars, and glycine betaine. For instance, Cui et al. [13] reported increased proline content and improved cellular water balance in wheat. In addition, melatonin supports protection against oxidative stress by stimulating the biosynthesis of secondary metabolites such as flavonoids, polyphenols, and anthocyanins, as demonstrated by Chen et al. [12] in grapevine.

Recent studies have reported that melatonin neutralizes ROS by upregulating antioxidant-related gene expression, thereby mitigating cellular damage [26], which contributed to improvements in SDW and FN, LDW and LL traits. These results align with Arnao & Hernández-Ruiz [8], who identified melatonin as a key regulator in stress tolerance.

Previous reports have confirmed that melatonin can enhance photosynthesis under stress conditions by inhibiting ROS activity and protecting chloroplasts [39, 40]. This effect was evident in the FC70 + M200 + CH100 treatment, which had the highest TChl concentration LDW in FC70 + M100 + CH100. Melatonin promotes photosynthetic efficiency by regulating the expression of photosynthesis-related genes such as rbcL and rbcS, thereby enhancing Rubisco activity and carbohydrate production. This supports vegetative growth (LDW, LL) and the translocation of nutrients to reproductive organs (SDW, FFW, FN) and storage tissues (TCW) [21]. This mechanism is consistent with Arnao et al. [7], who demonstrated that melatonin improves photosynthesis in drought-stressed plants.

Moreover, melatonin maintains osmotic balance by stimulating the synthesis of osmolytes such as proline. Our results indicated that melatonin-treated plants under FC70 exhibited better hydration status. Melatonin enhances proline synthesis by upregulating the expression of the P5CS gene and maintains turgor pressure, contributing to increased FFW in FC100 + M200 + CH200) and FN [4, 39]. These findings corroborate Imran et al. [21], who reported that melatonin enhances drought tolerance by modulating osmolyte accumulation.

Chitosan, as a biostimulant, mitigates the adverse effects of DS in plants by enhancing nutrient uptake, stimulating secondary metabolites, and reinforcing cellular stability [35]. The results of this study indicated that chitosan treatments at 100 and 150 mg L⁻¹, particularly under FC100 and FC70 conditions, improved plant performance. For example, the FC100 + M100 + CH100 treatment exhibited superior performance in SDW, TCW, and FN. It has been reported that chitosan enhances membrane permeability and stimulates root growth, thereby improving water and nutrient uptake, including nitrogen and potassium [33]. This effect was evident in the FC70 + M100 + CH150 treatment, which showed increased average corm weight and LDW. Chitosan enhances nitrogen metabolism by increasing nitrate reductase activity, facilitating nutrient translocation to both vegetative and reproductive organs, leading to increased LDW, SDW, and FN [28]. These results are consistent with Pichyangkura & Chadchawan [35], who demonstrated that chitosan improves nutrient uptake under stress conditions.

Furthermore, chitosan has been reported to activate defense signaling pathways such as jasmonic acid and stimulate the synthesis of secondary metabolites including crocin and flavonoids. This effect was observed in the FC100 + M200 + CH200 treatment, which exhibited higher SDW and FN. Chitosan upregulates the expression of the CsCCD2 gene, enhancing crocin biosynthesis and improving stigma quality [3, 9]. These findings align with Malerba & Cerana [28], who reported that chitosan enhances secondary metabolite production in stressed plants. Additionally, chitosan contributes to cellular stability by strengthening the cell wall and membrane integrity. By promoting lignin synthesis and membrane protein production, it reduces ROS-induced damage, aiding in the maintenance of RWC and vegetative growth traits such as LL and LDW [33, 38].

The combination of melatonin and chitosan exhibited synergistic effects in alleviating DS. Treatments consistently outperformed others across all measured traits (LDW, LL, SDW, FFW, FN, TCW, TChl, CAT, and RWC. It can be inferred that melatonin mitigated chlorophyll degradation, thereby preserving photosynthetic regulation and osmotic balance, while chitosan enhanced nutrient uptake, stimulated secondary metabolite production, and strengthened cell walls. Together, they jointly reduced oxidative and osmotic stress [21, 35]. Positive correlations between SDW and FFW (R² = 0.79 in the first year), FN (R² = 0.60 in the second year), and LL (R² = 0.78 in the second year) indicate a strong association between improved physiological processes and reproductive performance. Negative correlations of EL with LL (R² = −0.53) and FN (R² = −0.58) further confirm that membrane damage reduction by these treatments contributed to enhanced performance (Fig. 5A and B).

Conclusion

Our findings revealed that DS significantly limits saffron growth and productivity by reducing RWC and photosynthetic pigments, disrupting osmotic balance, and increasing oxidative stress. These adverse effects were particularly evident in the FC40 treatment, as demonstrated by reductions in LDW, LL, SDW, FFW, FN, TCW, and RWC. In contrast, the synergistic effects of melatonin and chitosan effectively mitigated these negative impacts through the regulation of photosynthetic processes, enhancement of the antioxidant defense system, maintenance of osmotic balance, and reinforcement of cellular stability. Notably, treatments involving low to moderate doses of melatonin and chitosan, particularly under FC100 and FC70 conditions, exhibited the most favorable outcomes. This two-year study, emphasizing the synergistic interaction between melatonin and chitosan, not only contributes to a deeper understanding of the physiological mechanisms in saffron under drought conditions but also offers practical and applicable strategies to enhance sustainable production under water-limited environments. The results of this research can provide valuable insights for farmers, researchers, and agricultural policymakers in optimizing irrigation management and utilizing biostimulants to improve saffron yield and quality in arid regions. Given the increasing global demand for saffron and the growing constraints on water resources, this study represents an important step toward sustainable agriculture and enhancing saffron resilience to climate change. Furthermore, future studies are recommended to focus on gene expression and metabolomics analyses to unravel the molecular mechanisms underlying the effects of these treatments more precisely. Due to its high economic value and pivotal role in the economy of arid regions, saffron requires innovative and efficient strategies to cope with environmental stresses.

Authors’ contributions

Sahar A. A. Malik Al-Saadi and Karzan Omer Qader design an experiment, Hatem Mohammed Hasan wrote the primilary draft of manuscript, Masoumeh Eskandari analysis of data and Heidar Meftahizade write the final manuscript, all editor study the final manuscript.

Funding

No specific financial credit was used in this experiment.

Data availability

Data is provided within the manuscript files.

Declarations

Ethics approval and consent to partcicipate

Not applicable.

Competing interests

The authors declare no competing interests.