Methyl jasmonate and ascorbic acid enhance salinity tolerance in pot marigold (Calendula officinalis L.) through improved morphophysiological and biochemical traits

Abstract

Salinity stress severely limits the growth and floral quality of ornamental plants in arid regions. This study evaluated the combined effects of foliar-applied methyl jasmonate (MeJA) and ascorbic acid (AA) on salinity tolerance in pot marigold (Calendula officinalis L.) under 8.64 dS m⁻¹ salinity stress. A factorial greenhouse experiment tested three MeJA concentrations (0, 100, 200 µM) and three AA concentrations (0, 100, 200 mg L⁻¹), with three replicates of six plants per treatment (18 plants total per treatment). The combined 200 µM MeJA and 200 mg L⁻¹ AA treatment significantly enhanced flower number (80%), diameter (37%), and longevity (33%), as well as shoot biomass (up to 50%) and root biomass (up to fourfold) compared to controls. It also reduced electrolyte leakage by 53% and increased relative water content (21%), photosynthetic efficiency (400%), and photosystem II performance (71.8%) relative to controls. Furthermore, antioxidant enzyme activities (catalase: 38.3%; peroxidase: 50%), proline accumulation (188.6%), and protein content (159.5%) increased compared to controls, reflecting enhanced osmotic regulation and cellular protection. These findings demonstrate that MeJA and AA synergistically improve salinity tolerance, boosting physiological resilience and floral quality under salt stress. Further field trials are needed to validate and optimize this approach for broader application.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16597-y.

Introduction

Soil salinity is a critical abiotic stressor that limits the growth and productivity of ornamental plants in arid regions, disrupting ionic homeostasis, inducing osmotic stress, and generating reactive oxygen species (ROS) that cause cellular damage, reduced photosynthetic efficiency, and inhibited biomass accumulation^1–3. Pot marigold (Calendula officinalis L.), a commercially valuable ornamental and medicinal species, faces significant challenges in saline soils due to its sensitivity to ionic imbalance and oxidative stress^4–6. Although pot marigolds possess inherent antioxidant systems, extreme salinity often overwhelms these defenses, necessitating innovative strategies to enhance stress resilience⁷. Phytohormones and antioxidants, such as methyl jasmonate (MeJA) and ascorbic acid (AA), have shown promise in mitigating salinity stress by enhancing antioxidant defenses and osmotic adjustment⁸. MeJA, a lipid-derived signaling molecule, upregulates antioxidant enzymes, glyoxalase systems, and photosynthetic machinery, reducing oxidative damage in salt-stressed plants^9,10. For instance, MeJA has been shown to lower electrolyte leakage and enhance photosynthetic efficiency under salinity in crops like rice¹⁰. Similarly, AA, a primary ROS scavenger, stabilizes cellular membranes and boosts antioxidant enzyme activity, improving salinity tolerance in halophytes like Limonium stocksii¹¹, barley (Hordeum vulgare)¹²pea (Pisum sativum)¹³.

Despite these advances, critical gaps remain in understanding the combined effects of MeJA and AA on salinity tolerance in pot marigold. While individual applications of MeJA and AA have been studied under salinity^9–13, and their combined effects have been explored under non-saline conditions in species like red willow¹⁴no research has investigated their synergistic potential in alleviating salt-induced oxidative and osmotic stress in pot marigold, particularly for improving floral traits and physiological resilience. This gap is significant, as synergy between MeJA and AA could offer a novel approach to enhance salinity tolerance by simultaneously targeting antioxidant defenses, osmotic adjustment, and photosynthetic performance.

This study hypothesizes that the co-application of MeJA and AA synergistically enhances pot marigold salinity tolerance by improving ion homeostasis, photosynthetic efficiency, and oxidative stress mitigation. The objective is to evaluate their individual and combined effects under 8.64 dS m⁻¹ salinity stress to develop a phytomanagement strategy for improving floral quality and plant resilience in saline environments.

Results

Effects of MeJA and AA on flower traits of pot marigold under saline stress

Two-way ANOVA revealed significant interactions between MeJA and AA affecting flower number (F = 6.79, p ≤ 0.01), flower diameter (F = 2.80, p ≤ 0.05), and flower longevity (F = 3.44, p ≤ 0.05) (Table 1). The highest flower number (18) was observed in the 200 µM MeJA + 100 mg/L AA treatment, an 80% increase compared to the control (10 flowers; Fig. 1a). Flower diameter reached a maximum of 72.59 mm in the 200 µM MeJA + 200 mg/L AA treatment, a 37% increase over the control (52.94 mm; Fig. 1b). Flower longevity peaked at 12.19 days in the same treatment, a 33% increase compared to the control (9.16 days; Fig. 1c).

Table 1.

Two-way ANOVA results showing the effects of Methyl jasmonate (MeJA) and ascorbic acid (AA) treatments on selected physiological traits of pot marigold (Calendula officinalis L.).

SOV	df	F value
		NF	FD	FL	EL	SFW	SDW	RFW	RDW
MeJA	2	30.9149 ***	12.5825 ***	17.1399***	92.0690 ***	20.0521 ***	5.7971 *	116.1390 ***	102.4566 ***
AA	2	9.3404 **	2.4055 ns	2.3322 ns	36.2411 ***	22.7935 ***	8.6380 **	111.7611 ***	65.6841 ***
MeJA × AA	4	6.7872 **	2.8016 *	3.4369 *	5.8293 *	7.4241 ***	4.3453 *	13.0690 ***	14.0199 ***
Error	18
Cv (%)		15.66	6.64	5.50	3.91	7.91	9.86	8.53	11.36

Significance levels: ns (not significant), *, **, *** indicate significance at p ≤ 0.05, 0.01, 0.001, respectively.

ND: Number of flowers; FD: Flower diameter; FL: Flower longevity; EL: Electrolyte leakage; SFW: Shoot fresh weight; SDW: Shoot dry weight; RFW: Root fresh weight; RDW: Root dry weight.

Fig. 1.

Effects of methyl jasmonate and ascorbic acid on flower number (a), flower diameter (b), flower longevity (c), electrolyte leakage (d), shoot fresh weight (e), shoot dry weight (f), root fresh weight (g), root dry weight (h) of pot marigold under saline soil conditions. Values are means of three replicates ± standard error (SE). Different letters above each bar indicate significant differences according to the Tukey’s HSD test (p ≤ 0.05).

Effects of MeJA and AA on physiological traits of pot marigold under saline stress

Two-way ANOVA indicated significant main and interaction effects of MeJA and AA on physiological traits. Significant MeJA × AA interactions were found for electrolyte leakage (F = 5.83, p ≤ 0.05), shoot fresh weight (F = 7.42, p ≤ 0.001), shoot dry weight (F = 4.35, p ≤ 0.05), root fresh weight (F = 13.07, p ≤ 0.001), and root dry weight (F = 14.02, p ≤ 0.001) (Table 1). Electrolyte leakage was lowest at 63.04% in the 100 µM MeJA + 200 mg/L AA treatment, a 53% reduction compared to the control (96.71%; Fig. 1d). Shoot fresh weight peaked at 90.97 g in the 200 mg/L AA treatment without MeJA, a 55% increase over the control (58.57 g; Fig. 1e). Shoot dry weight reached 11.25 g in the same treatment, a 53.5% increase compared to the control (7.33 g; Fig. 1f). Root fresh weight (51.97 g) and root dry weight (16.35 g) both peaked in the 200 µM MeJA + 200 mg/L AA treatment, showing approximately fourfold increases compared to the control (10.25 g and 3.12 g, respectively; Fig. 1g, h).

Effects of MeJA and AA on photosynthetic parameters of pot marigold under saline stress

Two-way ANOVA showed significant main and interaction effects of MeJA and AA on photosynthetic parameters. Significant MeJA × AA interactions were observed for net photosynthesis (F = 4.85, p ≤ 0.01), transpiration rate (F = 3.28, p ≤ 0.05), stomatal conductance (F = 7.31, p ≤ 0.01), intercellular CO₂ concentration (F = 11.40, p ≤ 0.001), water use efficiency (F = 6.99, p ≤ 0.01), and relative water content (RWC) (F = 4.17, p ≤ 0.05) (Table 2). Net photosynthesis peaked at 10.79 µmol CO₂ m⁻² s⁻¹ in the 200 µM MeJA + 200 mg/L AA treatment, a 400% increase compared to the control (2.16 µmol CO₂ m⁻² s⁻¹; Fig. 2a). Transpiration rate was lowest at 2.81 mmol H₂O m⁻² s⁻¹ in the same treatment, a 55% reduction from the control (6.27 mmol H₂O m⁻² s⁻¹; Fig. 2b). Stomatal conductance (0.17 mmol m⁻² s⁻¹) and intercellular CO₂ concentration (156.6 µmol mol⁻¹) also peaked in this treatment, increasing by 183% and 62.4% compared to the control (0.06 mmol m⁻² s⁻¹ and 96.42 µmol mol⁻¹, respectively; Fig. 2c, d). Water use efficiency (2.33 µmol CO₂ mmol⁻¹ H₂O) and RWC (66.67%) were highest in this treatment, with increases of 228% and 21% over the control (0.71 µmol CO₂ mmol⁻¹ H₂O and 55%, respectively; Figs. 2e, f).

Table 2.

Two-way ANOVA results showing the effects of Methyl jasmonate (MeJA) and ascorbic acid (AA) on photosynthetic parameters and chlorophyll fluorescence characteristics traits of pot marigold (Calendula officinalis L.).

SOV	df	F value
		Pn	E	gs	Ci	WUE	RWC	FO	Fm	Fv/Fm	ΦPSII
MeJA	2	76.7904 ***	26.3083 ***	17.6462 ***	319.8012 ***	108.5625 ***	40.6528 ***	19.1011 ***	79.9823 ***	50.1034 ***	37.1232 ***
AA	2	37.2403 ***	57.9432 ***	26.2769 ***	94.5239 ***	43.2702 ***	7.5851 **	22.1330 ***	6.4956 **	29.2069 ***	7.1232 **
MeJA × AA	4	4.8522 **	3.2843 *	7.3077 **	11.3950 ***	6.9897 **	4.1747 *	19.2128 ***	28.5414 ***	31.6896 ***	11.6127 ***
Error	18
Cv (%)		13.32	10.44	10.88	2.23	9.96	2.96	6.18	2.32	1.42	19.30

Significance levels: ns (not significant), *, **, *** indicate significance at p ≤ 0.05, 0.01, 0.001, respectively.

Pn: Net photosynthetic rate; E: Transpiration rate; gs: Stomatal conductance; Ci: Intercellular CO₂ concentration; WUE: Water use efficiency; RWC: Relative water content; F_o: minimum fluorescence; F_m: maximum fluorescence; F_v/F_m: maximum quantum yield of PSII; ΦPSII: effective quantum yield of PSII.

Fig. 2.

Effects of methyl jasmonate and ascorbic acid on net photosynthetic rate; Pn (a), transpiration rate: E (b); stomatal conductance: gs (c); intercellular CO₂ concentration: Ci (d), water use efficiency: WUE (e), and relative water content: RWC (f) of pot marigold under saline soil conditions. Values are means of three replicates ± standard error (SE). Different letters above each bar indicate significant differences according to the Tukey’s HSD test (p ≤ 0.05).

Effects of MeJA and AA on chlorophyll fluorescence of pot marigold under saline stress

Two-way ANOVA revealed significant main and interaction effects of MeJA and AA on chlorophyll fluorescence parameters. Significant MeJA × AA interactions were detected for F_o (F = 19.21, p ≤ 0.001), F_m (F = 28.54, p ≤ 0.001), F_v/F_m (F = 31.69, p ≤ 0.001), and ΦPSII (F = 11.61, p ≤ 0.001) (Table 2). The lowest F_o (82.67) was recorded in the 200 mg/L AA treatment without MeJA, a 25.4% reduction compared to the control (103.67; Fig. 3a). The highest F_m (380.67) was observed in the 200 µM MeJA treatment without AA, a 30.8% increase over the control (291.10; Fig. 3b). F_v/F_m peaked at 0.77 in the 200 mg/L AA treatment without MeJA, a 30.5% increase compared to the control (0.59; Fig. 3c). ΦPSII reached its maximum of 0.67 in the 200 µM MeJA + 200 mg/L AA treatment, a 71.8% increase compared to the control (0.39; Fig. 3d).

Fig. 3.

Effects of methyl jasmonate and ascorbic acid on minimum fluorescence: F_o (a), maximum fluorescence: F_m (b), maximum quantum yield of PSII F_v/F_m (c), effective quantum yield of PSII: ΦPSII (d), chlorophyll a (e), chlorophyll b (f), total chlorophyll (g), and total carotenoids (h) of pot marigold under saline soil conditions. Values are means of three replicates ± standard error (SE). Different letters above each bar indicate significant differences according to the Tukey’s HSD test (p ≤ 0.05).

Effects of MeJA and AA on photosynthetic pigments of pot marigold under saline stress

Two-way ANOVA showed significant main and interaction effects of MeJA and AA on photosynthetic pigments. Significant MeJA × AA interactions were observed for chlorophyll a (F = 20.21, p ≤ 0.001), chlorophyll b (F = 6.92, p ≤ 0.01), total chlorophyll (F = 13.42, p ≤ 0.001), and carotenoids (F = 4.68, p ≤ 0.01). However, the main effect of AA on carotenoids was not significant (F = 2.53, p > 0.05) (Table 3). Chlorophyll a peaked at 1.23 mg g⁻¹ FW in the 200 µM MeJA + 200 mg/L AA treatment, a 44.7% increase compared to the control (0.85 mg g⁻¹ FW; Fig. 3e). Chlorophyll b (0.73 mg g⁻¹ FW), total chlorophyll (2.13 mg g⁻¹ FW), and carotenoids (0.31 mg g⁻¹ FW) also peaked in this treatment, with increases of 160%, 71.7%, and 34.7% compared to the control (0.28 mg g⁻¹ FW, 1.24 mg g⁻¹ FW, and 0.23 mg g⁻¹ FW, respectively; Fig. 3f–h).

Table 3.

Two-way ANOVA results showing the effects of Methyl jasmonate (MeJA) and ascorbic acid (AA) on photosynthetic pigment contents and biochemical parameters of pot marigold (Calendula officinalis L.).

SOV	df	F value
		Chl a	Chl b	Chl T	Car	CAT	POD	TSP	PC
MeJA	2	27.8595 ***	50.7675 ***	41.7956 ***	18.6724 ***	0.8099 ns	17.6471 ***	24.5312 ***	49.9362***
AA	2	12.1588 ***	10.8948 ***	11.5994 ***	2.5345 ns	4.9583 *	3.3529 ns	10.0976 **	20.1232 ***
MeJA × AA	4	20.2117 ***	6.9247 **	13.4177 ***	4.6810 **	5.6044 **	3.5294 *	9.6733 ***	3.3831 *
Error	18
Cv (%)		3.97	10.20	5.61	7.87	13.06	19.30	11.32	13.63

Significance levels: ns (not significant), *, **, *** indicate significance at p ≤ 0.05, 0.01, 0.001, respectively.

Chl a: chlorophyll a; Chl b: chlorophyll b; Chl T: total chlorophyll; Car: total carotenoids; CAT: catalase activity; POD: peroxidase activity; TSP: total soluble protein; PC: proline content.

Effects of MeJA and AA on biochemical parameters of pot marigold under saline stress

Two-way ANOVA indicated significant main and interaction effects of MeJA and AA on biochemical parameters. Significant MeJA × AA interactions were found for catalase activity (F = 5.60, p ≤ 0.01), peroxidase activity (F = 3.53, p ≤ 0.05), total soluble protein (F = 9.67, p ≤ 0.001), and proline content (F = 3.38, p ≤ 0.05) (Table 3). Catalase activity peaked at 0.65 U mg⁻¹ protein in the 200 µM MeJA + 200 mg/L AA treatment, a 38.3% increase compared to the control (0.47 U mg⁻¹ protein; Fig. 4a). Peroxidase activity reached 0.06 U mg⁻¹ protein in the same treatment, a 50% increase compared to the control (0.04 U mg⁻¹ protein; Fig. 4b). Total soluble protein was highest at 389.83 µg g⁻¹ FW in the 200 µM MeJA treatment without AA, a 159.6% increase compared to the control (150.17 µg g⁻¹ FW; Fig. 4c). Proline content peaked at 4.30 µmol g⁻¹ FW in the 200 µM MeJA + 200 mg/L AA treatment, a 188.6% increase compared to the control (1.49 µmol g⁻¹ FW; Fig. 4d).

Fig. 4.

Effects of methyl jasmonate and ascorbic acid on catalase activity: CAT (a), peroxidase activity: POD (b), total soluble protein (c), and proline content (d) of pot marigold under saline soil conditions. Values are means of three replicates ± standard error (SE). Different letters above each bar indicate significant differences according to the Tukey’s HSD test (p ≤ 0.05).

Discussion

Effects of MeJA and AA on flower traits of pot marigold under saline stress

The combined application of 200 µM MeJA and 200 mg/L AA significantly increased flower number by 80%, flower diameter by 37%, and flower longevity by 33% in pot marigold (Calendula officinalis L.) under 8.64 dS m⁻¹ salinity stress (Fig. 1a–c). These improvements align with findings in summer savory¹⁵, which demonstrated enhanced flower number and essential oil yield under drought stress, although flower diameter and longevity were not evaluated in that study. MeJA also mitigated salinity stress in purple basil (Ocimum basilicum L.)¹⁶ and narcissus¹⁷, improving flower quality and longevity, but these studies did not quantify flower diameter. Moreover, AA (200 mg L⁻¹) doubled lisianthus (Eustoma grandiflorum L.) vase life by stabilizing cell membranes and reducing water loss¹⁸. In anthurium, AA treatments reduced chilling injury and extended vase life during cold storage¹⁹. In chrysanthemum, AA maintained petal turgor and chlorophyll content two days longer than controls²⁰. Despite differing life cycles, AA’s enhancement of flower longevity via ROS scavenging and turgor maintenance is likely applicable to pot marigold, given their shared physiological responses to oxidative stress¹³. The synergistic of MeJA and AA promotes floral development by enhancing cell expansion, nutrient uptake, and cellular integrity, mitigating salinity-induced stress.

Effects of MeJA and AA on membrane stability of pot marigold under saline stress

The 100 µM MeJA + 200 mg/L AA treatment significantly reduced electrolyte leakage by 53% (Fig. 1d), indicating enhanced membrane stability. MeJA reduced electrolyte leakage by 7.8–35% in perennial ryegrass²¹ and peppermint²² under salt stress by upregulating antioxidant enzymes that mitigate oxidative damage to membrane lipids. AA directly contributes to this reduction by scavenging ROS that damage membrane lipids, as shown in pea¹³, where AA stabilized membranes under 100 mM NaCl, and in barley¹², where AA reduced ion leakage by enhancing redox homeostasis. This synergistic action of MeJA and AA minimizes ion leakage by protecting membrane integrity and reducing oxidative stress, as evidenced by lower malondialdehyde levels²³. These mechanisms collectively enhance cellular stability, enabling pot marigold to maintain physiological functions under salinity stress.

Effects of MeJA and AA on fresh and dry weight of shoots and roots of pot marigold under saline stress

Treatment with 200 µM MeJA + 200 mg/L AA increased fresh and dry weights of shoots by over 50% and roots by approximately fourfold (Figs. 1e–h). MeJA promotes growth and alleviates stress-induced damage through enhanced nutrient uptake and osmolyte accumulation (e.g., proline, increased by 188.6% in our study; Fig. 4d), as observed in maize under drought stress²⁴ and Cosmos bipinnatus under cadmium stress²⁵. Combined MeJA and AA treatments in red willow increased leaf production, boosting biomass by mitigating oxidative damage¹⁴. This synergy supports cellular expansion and metabolic activity under salinity stress.

Effects of MeJA and AA on photosynthetic parameters of pot marigold under saline stress

The 200 µM MeJA + 200 mg/L AA treatment enhanced net photosynthesis by 400%, stomatal conductance by 183%, intercellular CO₂ concentration by 62.4%, and water use efficiency by 228% (Figs. 2a–e). MeJA protects photosynthesis in wheat²⁶ and polyethylene glycol-treated plants²⁷, though dose-dependent reductions were noted in soybean²⁸. The treatment also improved relative water content by 21% (66.67% vs. 55%; Fig. 2f), consistent with MeJA’s role in maintaining leaf water content in summer savory¹⁵ and peppermint²⁹. MeJA can delay plant dehydration by enhancing osmolyte accumulation and stomatal regulation, as demonstrated in strawberry, where it increased relative water content and reduced water loss^30,31. These effects, combined with AA’s role in mitigating photoinhibition, support photosynthetic efficiency under salinity stress.

Effects of MeJA and AA on chlorophyll fluorescence of pot marigold under saline stress

The 200 µM MeJA + 200 mg/L AA treatment increased the effective quantum yield of photosystem II (ΦPSII) by 71.8% (Fig. 3d) and maximum quantum yield (F_v/F_m) by 30.5% (Fig. 3c). MeJA enhances PSII efficiency by stabilizing the D1 protein and reducing photoinhibition under abiotic stresses, as observed in heat-stressed wheat³², drought-stressed pepper (Capsicum annuum L.)³³, and salinity-stressed saffron (Crocus sativus L.)³⁴. In maize, MeJA upregulated carotenoid biosynthesis, enhancing photoprotection and electron transport efficiency³⁵. AA mitigates photoinhibition by scavenging ROS in chloroplasts, as reported in pea under salinity, where it enhanced ΦPSII and maintained photosynthetic activity¹³. This synergy improves light utilization and electron transport, supporting photochemical efficiency and stress resilience in pot marigold.

Effects of MeJA and AA on photosynthetic pigments of pot marigold under saline stress

The 200 µM MeJA + 200 mg/L AA treatment significantly increased chlorophyll a by 44.7%, chlorophyll b by 160%, total chlorophyll by 71.7%, and carotenoids by 34.7% (Figs. 3e–h). MeJA protects chloroplasts by upregulating antioxidant enzymes and stabilizing photosynthesis-related proteins, as observed in Crithmum maritimum³⁶, where it mitigated salinity-induced chlorophyll degradation. Similarly, in maize, MeJA enhanced carotenoid biosynthesis, supporting photoprotection³⁵. AA is recognized for maintaining chlorophyll content, as evidenced by our data showing a 71.7% increase in total chlorophyll and in pea¹³, where AA increased chlorophyll a by 41.1% and chlorophyll b by 56.1% under 100 mM NaCl by scavenging ROS and reducing lipid peroxidation. This aligns with findings in barley, where AA improved chlorophyll stability under salinity stress, enhancing photosynthetic capacity¹². The synergistic action of MeJA and AA preserves photosynthetic pigments by mitigating oxidative damage to chloroplast membranes, maintaining electron transport efficiency, and supporting photochemical reactions under salinity stress. These enhancements are critical for sustaining photosynthesis and overall plant resilience in saline environments.

Effects of MeJA and AA on antioxidant defense and biochemical responses of pot marigold under saline stress

This study demonstrated that MeJA and AA, particularly at 200 µM MeJA + 200 mg/L AA, significantly enhanced antioxidant defense systems, total protein content, and proline levels in pot marigold under salinity stress (Figs. 4a-d). The results of this study are aligning with findings that MeJA reduces ROS accumulation under salinity³⁶, drought²⁴, heat³⁷ and cod³⁸ stress by enhancing antioxidant enzyme activities³⁹. MeJA enhanced photosynthetic capacity and nitrogen uptake, promoting plant health in wheat under heat stress⁴⁰ and in grapevines under drought stress⁴¹. Synergistic effects with antioxidants like melatonin further amplified antioxidant enzyme activities, reducing oxidative stress more effectively⁴⁰. AA, a key non-enzymatic antioxidant, directly scavenged ROS and supported APX activity, maintaining redox homeostasis and complementing MeJA’s effects^24,40. Consistent with our findings, MeJA treatment increased proline content in Anchusa italica under salinity stress⁴² and in maize under drought stress²⁴ which correlated with enhanced relative water content and antioxidant activity. MeJA’s mechanism involves osmotic regulation, membrane protection, and ROS scavenging, while also elevating AA levels to enhance stress tolerance^23,39.

Mechanistic insights and limitations

MeJA activates jasmonic acid signaling through COI1 receptor binding, promoting MYC2-mediated expression of genes involved in antioxidant enzyme activity (e.g., catalase, peroxidase) and osmolyte biosynthesis (e.g., proline synthase)^23,43, as seen in Arabidopsis⁹. This pathway reduces ROS accumulation and enhances osmotic adjustment, critical for salinity tolerance. AA scavenges ROS and supports ascorbate-glutathione cycle activity, stabilizing redox-sensitive components like MYC2, as reported in pea¹³. Crosstalk between jasmonic acid and abscisic acid signaling, mediated by MYC2 and ABI5 transcription factors, upregulates NAC family genes (e.g., ANAC019) and osmotic stress-responsive genes (e.g., RD29A), promoting water retention^44–46. MeJA-induced heat shock proteins (e.g., HSP70) and NAC transcription factors further support protein stability, as seen in maize⁴⁷, while AA ensures proper protein folding through redox buffering⁴⁸.

However, the study’s limitations include the absence of a salinity-free control, limiting baseline comparisons, and the use of greenhouse conditions, which may not reflect field variability. Testing a single cultivar in one season (autumn 2024) and evaluating only two doses of MeJA and AA restrict generalizability and dose-response insights. Additionally, the study did not assess long-term effects or economic feasibility of MeJA and AA applications. Future research should incorporate salinity-free controls, field trials, diverse cultivars, multi-season studies, broader dose ranges, and cost-benefit analyses to optimize application protocols and enhance practical applicability.

Conclusion

This study demonstrates that exogenous application of MeJA and AA, individually and in combination, significantly enhances growth, physiological, and biochemical responses of pot marigold under saline soil conditions (8.64 dS m⁻¹). The treatments improved flower number, diameter, longevity, and biomass of roots and shoots, with the combined 200 µM MeJA + 200 mg/L AA treatment producing the most pronounced benefits. Physiological and biochemical enhancements included increased chlorophyll content, photosynthetic efficiency, relative water content, antioxidant enzyme activities, and osmoprotectant accumulation, alongside reduced electrolyte leakage and oxidative stress, highlighting a synergistic enhancement of salt tolerance. However, given the study’s controlled environment, single-cultivar focus, and variability in some traits, further research is required. Future work should prioritize field trials across diverse cultivars and environmental conditions to validate these findings and optimize application strategies. Optimizing application methods (e.g., foliar sprays), dosages, and timing tailored to specific crops and local conditions will be critical. Integration of MeJA and AA treatments with current horticultural practices such as fertilization and irrigation, as well as consideration of economic feasibility and material availability, are essential for practical adoption by farmers and breeders. Overall, these preliminary results provide a promising approach for improving salinity tolerance in ornamental and medicinal plants, supporting sustainable horticultural production.

Materials and methods

Plant material and experimental design

The experiment was conducted in autumn 2024 under natural daylight conditions in a research greenhouse at Shahid Bahonar University of Kerman, Iran (30°N, 57°E, 1754 m altitude), with no supplemental lighting. The average photosynthetic photon flux density (PPFD) was approximately 600–800 µmol m⁻² s⁻¹ during the 16-hour photoperiod. The autumn season ensured stable temperatures (20.5 °C) and relative humidity (20–25%), minimizing seasonal fluctuations that could influence plant responses. The soil mixture used consisted of sand, clay, and decomposed manure in a ratio of 2:1:1 by volume, with an electrical conductivity of 8.64 dS m⁻¹. Seedlings of pot marigold (Calendula officinalis L.) were transplanted into pots (dimensions: 25.5 cm diameter and 25 cm height), with one plant per pot after establishment; each pot contained one plant, which served as the experimental unit for all measurements. A nutrient solution containing 10% nitrogen (N), 8% phosphorus (P), 4% potassium (K), 0.1% iron (Fe), and 0.1% zinc (Zn) was applied through irrigation.

The experiment was designed as a completely randomized design (CRD) to evaluate the interactive effects of methyl jasmonate (MeJA) and ascorbic acid (AA) on pot marigold under salinity stress (8.64 dS m⁻¹). Two factors were tested: MeJA at three levels (0, 100, and 200 µM) and AA at three levels (0, 100, and 200 mg/L), resulting in a 3 × 3 factorial arrangement with 9 treatment combinations. Each treatment was conducted with three independent replicates, each containing six individual plants (pots), yielding 18 plants per treatment (3 replicates × 6 plants per replicate). The total number of experimental units was 9 treatments × 18 plants = 162 plants. Pots were assigned treatments randomly in the greenhouse to ensure a completely randomized design and minimize positional effects. Data from the six plants per replicate were averaged to obtain a single value per replicate for statistical analysis.

Foliar applications of MeJA and AA were performed three times during the experiment: five weeks after transplantation, at the 4–6 leaf stage, and then at two-week intervals. Each plant was sprayed with 50 mL of the respective solution (containing MeJA and AA in deionized water with 0.1% Tween-20 as a surfactant) until runoff on the leaves. Spraying was done in the morning between 8:00 and 9:00 AM under stable greenhouse conditions (temperature 20–22 °C, relative humidity 20–25%, and light intensity 400–600 µmol m⁻² s⁻¹) to ensure optimal absorption and minimal environmental variability. The control group consisted of plants grown under saline conditions (8.64 dS m⁻¹) without MeJA and AA application; these were sprayed only with deionized water containing 0.1% Tween-20 following the same spraying schedule and conditions as the treatments. A salinity-free control group was not included because the study’s objective was to evaluate the effectiveness of MeJA and AA in alleviating salinity stress.

Morphological, physiological, and biochemical trait measurement

Sample collection

Morphological traits (flower number, diameter, and longevity) were assessed throughout the flowering period, with daily counts of flower buds to quantify total flower number, aligning with the plant’s reproductive phase. Physiological and biochemical measurements, including electrolyte leakage, photosynthetic gas exchange, chlorophyll fluorescence, photosynthetic pigments, enzymatic activities (catalase and peroxidase), total soluble protein, and proline content, were conducted 7 days after the final foliar application of MeJA and AA, at the peak flowering stage of pot marigold, to capture stabilized treatment effects under salinity stress. Shoot and root fresh and dry weights were measured at the end of the experiment during plant harvesting, when plants were uprooted.

Flower number, flower diameter, flower longevity

To quantify the total number of flowers, daily counts of flower buds were conducted after each blooming event. A cumulative count was maintained throughout the experimental period to determine the total number of flowers produced⁴⁹. Flower diameter was measured on fully open flowers to ensure consistency. For each plant, three flowers were randomly selected and measured. The diameter was recorded as the longest distance across the flower, passing through the center, using a digital caliper with an accuracy of 0.01 mm⁵⁰. The duration of flower longevity was determined from the moment the petals began to open slightly, revealing the stamens, until they wilted or dropped off. For each replication, 10–12 newly emerging floral buds were randomly selected from 3 to 5 plants. Throughout the flowering season, the dates of opening and wilting for each flower were meticulously recorded to assess the longevity of individual flowers⁵¹.

Electrolyte leakage

Electrolyte leakage was measured to evaluate the stability of cell membranes under the given experimental conditions⁵². Fresh leaf samples weighing about 0.5 g were collected from each plant and gently washed with deionized water to remove any electrolytes present on the surface. The leaves were then cut into 1 cm pieces and placed into test tubes containing 10 mL of deionized water. These tubes were kept at room temperature (25 °C) with gentle shaking for 24 h to allow electrolytes to diffuse from damaged cells. The initial electrical conductivity (EC₁) of the solution was recorded using a conductivity meter (Apera EC20, Apera Instruments, China). Subsequently, the samples were autoclaved at 121 °C for 20 min to release all remaining electrolytes. After cooling, the final electrical conductivity (EC₂) was measured. The percentage of electrolyte leakage was calculated using the following formula:

Fresh and dry weights of aerial and root parts

Plants were carefully removed from pots, and soil around the roots was gently washed off. Shoots and roots were separated, and their fresh weights were measured using a precision scale. Samples were dried in an oven at 70 °C until a constant weight was achieved, after which dry weights were recorded⁵³.

Photosynthetic gas exchange measurements

Photosynthetic gas exchange parameters were measured using a portable infrared gas analyzer (IRGA) system (LI-COR LI-6800, LI-COR Biosciences, Lincoln, NE, USA). Fully expanded leaves were selected and allowed to acclimate to ambient conditions for 30 min prior to measurement. Under controlled environmental conditions—light intensity set at 1,500 µmol photons m⁻² s⁻¹, CO₂ concentration maintained at 400 ppm, and leaf temperature regulated at 25 °C—key physiological parameters were recorded, including: net photosynthetic rate (Pn) expressed in µmol CO₂ m⁻² s⁻¹, stomatal conductance (gs) measured in mmol H₂O m⁻² s⁻¹, intercellular CO₂ concentration (Ci) in µmol mol⁻¹, transpiration rate (E) expressed in mmol H₂O m⁻² s⁻¹, and water use efficiency (WUE) in µmol CO₂ mmol⁻¹ H₂O. Measurements were performed between 9:00 AM and 12:00 PM to reduce variability caused by diurnal changes⁵⁴.

Relative water content (RWC)

RWC was measured to evaluate the water status of plant leaves⁵⁵. Fresh, fully expanded leaves were collected and immediately weighed to determine their fresh weight (F_W). The leaves were then submerged in distilled water and kept in the dark at room temperature for several hours until full turgidity was reached. After carefully blotting surface moisture, the turgid weight (T_W) was recorded. Subsequently, the samples were dried in an oven at 70 °C until a constant weight was obtained to measure the dry weight (D_W). The RWC was calculated using the formula:

Chlorophyll fluorescence analysis

Photosystem II (PSII) efficiency was measured using a pulse-amplitude modulation (PAM) fluorometer (Junior_PAM, WALZ, Germany). Fully expanded leaves were dark-adapted for 30 min to allow photosynthetic apparatus relaxation before exposure to a saturating light pulse of 3,000 µmol photons m⁻² s⁻¹. This procedure enabled the determination of the maximum quantum yield of PSII (F_v/F_m), calculated as (F_m − F_o)/F_m, where F_o is the minimum fluorescence of dark-adapted leaves and Fm is the maximum fluorescence following the saturating pulse. Additionally, the effective quantum yield of PSII (ΦPSII) was recorded under actinic light to evaluate the operational efficiency of PSII during photosynthesis⁵⁶.

Spectrophotometric chlorophyll quantification

Fresh leaf samples were homogenized in 70% acetone and centrifuged at 350 × g for 15 min at room temperature to obtain a clear extract. The absorbance of the supernatant was measured using a spectrophotometer at wavelengths 663.2 nm, 646.8 nm, and 470 nm. Pigment concentrations were calculated using the following Eq. ⁵⁸, expressed as milligrams per gram of fresh leaf weight (mg g⁻¹ FW):

Preparation of enzyme extracts and total soluble protein

Fresh leaves weighing 0.5 g were collected and immediately ground in 5 mL of ice-cold 50 mM potassium phosphate buffer (pH 7.0) using a chilled mortar and pestle. The homogenate was then centrifuged at 12,000 × g for 15 min at 4 °C. The resulting supernatant, referred to as the “enzyme extract,” contains soluble enzymes and was used for enzyme activity assays and protein quantification. Enzyme extracts were analyzed immediately or stored on ice at 4 °C for no longer than 2 h.

Catalase (CAT) activity assay

Catalase (CAT) activity was determined spectrophotometrically by measuring the decomposition of H₂O₂ at 240 nm according to the method of Aebi⁵⁸. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, and enzyme extract. The decrease in absorbance at 240 nm was monitored for 2 min. One unit (U) of CAT activity is defined as the amount of enzyme that decomposes 1 µmol of H₂O₂ per minute per mg of protein, expressed as U mg⁻¹ protein, where 1 U equal 1 µmol H₂O₂ decomposed per minute under assay conditions (pH 7.0, 25 °C).

Peroxidase (POD) activity assay

Peroxidase (POD) activity was assayed by monitoring the oxidation of guaiacol in the presence of H₂O₂ at 470 nm as described by Chance and Maehly⁵⁹. The assay mixture included 50 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, 10 mM H₂O₂, and enzyme extract. The increase in absorbance at 470 nm was recorded for 3 min. One unit (U) of POD activity is defined as the amount of enzyme causing an absorbance increase of 0.01 at 470 nm per minute per mg of protein, expressed as U mg⁻¹ protein, where 1 U equals an absorbance increase of 0.01 at 470 nm per minute under standard assay conditions (pH 7.0, 25 °C).

Total soluble protein

Protein concentration in the enzyme extracts was measured using the Bradford assay⁶⁰. In brief, 100 µL of the enzyme extract was mixed with 5 mL of Bradford reagent and incubated at room temperature for 10 min. Absorbance was read at 595 nm, and protein levels were calculated from a standard curve prepared with bovine serum albumin (BSA).

Proline content

Free proline content in leaf tissues was quantified using the spectrophotometric⁶¹. Fresh leaf samples (0.5 g) were homogenized in 10 mL of 3% sulfosalicylic acid and centrifuged at 3,500 × g for 10 min to pellet debris. The supernatant (2 mL) was mixed with 2 mL of acid ninhydrin reagent and 2 mL of glacial acetic acid, then heated in a boiling water bath at 100 °C for 1 h. After cooling in ice, 4 mL of toluene was added, and the mixture was vortexed for 30 s using an Orbital MX-F Vortex Mixer (Drawell Scientific Instrument Co., Ltd., China) at a fixed speed of 3000 rpm to ensure thorough mixing. The absorbance of the supernatant was measured at 520 nm. Proline concentration was determined using a standard curve of L-proline and expressed as µmol per gram fresh weight (µmol g⁻¹ FW).

Statistical analysis

The experiment was conducted with three replicates, each including 6 individual plants (pots), in a factorial design with 9 treatment combinations (3 MeJA × 3 AA), totaling 18 plants per treatment. Data from the 6 plants per replicate were averaged to obtain a single value per replicate. Two-way analysis of variance (ANOVA) was performed using SAS version 9.4. Mean comparisons were conducted using Tukey’s Honestly Significant Difference (HSD) test at p ≤ 0.05. Results are presented as mean ± standard error (SE), with ANOVA results summarized in Tables 1, 2 and 3.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

A.L. Methodology, Data curation, carried out the experiment; S.S. Supervision, Formal analysis, Writing– review & editing; V.R.S. Supervision, designed the research. All authors reviewed the manuscript.

Funding

Not applicable.

Data availability

Data is provided within the supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

We confirm that all the experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. All of the material is owned by the authors and/or no permissions are required.