Ameliorative effect of melatonin on different tomato genotypes to induce heat stress tolerance by modulating growth and physiological attributes

2.1 Experimental design

A pot trial was performed under two factor factorial randomized complete block design (RCBD) to evaluate the heat stress-mitigation effect of melatonin spray in four tomato cultivars. Tomato seeds of four cultivars (two heat tolerantand two heat sensitive) were sown. Pots of all tomato cultivars were placed in field under sunshine in order to face heat stress by plant. Two treatments were applied [control (without foilarspray) and melatonin @ 25 µM)] to all genotypes of tomato and each was replicated five times. Heat tolerant genotypes were included as T60 F1 and Supercash F1 while heat sensitive genotypes were named as Nagina and Naqeeb.

The seeds of tested genotypes of tomato were grown in 12-inch plastic pots filled with peat moss for growth medium. Half-strength Hoagland and Arnon (1950) nutrient solution, was applied as a source of nutrients. Pots were placed in the field of research area. The variations in climate of Sargodha city remained 41/5 °C and rainfall of 115/5 mm Whereas ideal temperature for tomato growth varies between 22–28 °C. Melatonin was applied at two growth stages of tomato i. one week before the maturity stage. ii. One week before the harvesting stage and following parameters were studied.

2.2 Growth parameters measurements

Data regarding growth attributes was collected at the end of stress, 40 days after the sowing of tomato seeds. Leaf samples and plant samples of individual replications were uprooted and washed with distilled water and attached particles of growing media were removed from the roots. After that plants blotted with filter paper for the removal of water present on leaves and roots.

Plant fresh biomass, plant dry biomass, leaf area, stem girth, photosynthesis rate (Pn), stomatal conductance, transpiration rate (E), water use efficiency (Y/Et), leaf temperature, chlorophyll contents, electrolyte leakage (EL), hydrogen peroxide (H₂O₂), soluble protein contents and number of viable seeds were observed according to standard procedures. Leaf area of plant was measured with the help of Leaf area meter (Ll-3100; LI-COR Inc., USA).

2.3 Physiological measurements

From top fully expanded 2nd leaf of a young plant from each replication of treatments was selected to record the photosynthetic rate, transpiration rate, and stomatal conductance with the help of photosynthesis measuring-system CI-340 transportable infrared gas analyzer (Analytical Development Company, Hoddesdon, England). This information was recorded between 10.00 and 12.00 A.M. of the day with subsequent modifications: atmospheric pressure 99.9 kPa, molar flow of air, per unit area of leaf 403.3 mmol m⁻² S^-1, water vapor pressure into chamber ranged from 6.0 to 8.9 m bar, leaf temperature ranged from 28.4 − 32.4 °C, ambient CO₂ concentration 352 mol mol⁻¹, PAR at surface of leaves was highest upto 1711 mol m⁻² S^-1 and ambient temperature was ranged from 22.4-27.9 °C.

Chlorophyll content meter (CCM-300, ADC Bioscientific, UK) was used to estimate chlorophyll contents from fully matured leaf samples of tomato plants. Water use efficiency was measured by using the following equation. $$\mathit{WUE}=\frac{Y}{\mathrm{E}\mathrm{t}}$$

Where, Y=Crop yield and Et = Evapotranspiration rate.

2.4 Electrolyte leakage

Electrolyte leakage (EL) measures cell damage. It was determined by the method of Shi et al. (2006). 0.25 g leaf sample was kept in 25 ml deionized water for 24 h in test tubes. After 24 h time period, EC1 was measured by using electrical conductivity meter. Then test tubes were placed in water bath for 1 h at 90 ℃. After that, EC2 was measured again. Thus EL% was the determined by following equation.. EC₁ / EC₂ x 100.

2.5 H₂O₂ quantification

Analysis of hydrogen peroxide (H₂O₂) quantification was made by a method described by MacNevin and Urone (1953) with some modifications (Brennan and Frenkel, 1977; Rivero et al., 2007).

2.6 Leaf temperature

Leaf temperature was estimated by an Infrared thermometer (AmiciKart® Digital Laser IR Infrared Thermometer-GM320) from 11 am to 12 noon during the stress period.

2.7 Protein contents

The soluble protein contents were estimated through Bradford (1976) method using spectrophotometer UV (PG instrument T60).

2.8 Number of viable seeds per fruit

To measure the number of viable seeds, three fruits per plant were randomly selected and their seeds were counted manually by gently extracting seeds from tomato fruit through manual process and their viability was tested in petri dishes using Whatman filter paper wetted with double distilled water. The seeds that ruptured their seed coat and showed growth of their cotyledon were counted and considered viable seeds.

2.9 Enzyme assays

Activity of enzymatic antioxidants including NOS, GR, NR and GST was assayed by using their respective detection kits (Solarbio Life science, Beijing, China) according to protocol. Centrifugation rate and molar extinction was different for all determined enzymes. These kits were used after digestion and collection of supernatant solution of 0.1 g leaf sample.

2.10 Experimental design and statistical analysis

The data was analyzed through statistical methods illustrated by Gomez and Gomez (1984). To find the difference of significance between treatment means at P<0.05 (n = 5) Tukey HSD test was used. Data were evaluated through Statistix 8.1 software.

3.1 Impact of melatonin foliar treatment on morphological attributes of tomato genotypes

The results (Table 1) showed that under the foliar application of melatonin, plant fresh biomass was increased as 41.62 % and 40.98 % in heat tolerant (T60 F1 and Super cash F1) genotypes of tomato respectively showing values as 651.99 g and 650.56 g of tomato when compared to their respective control. Similarly, fresh biomass in heat sensitive (Nagina and Naqeeb) genotypes was recorded as 473.09 g and 472.01 g respectively showing 34.11 % and 31.20 % increase than their respective control. However, all genotypes either heat tolerant or heat sensitive were increased significantly (p ≤ 0.05) under melatonin spray. However, heat tolerant genotypes of tomato were more responsive than heat-sensitive cultivars of tomato.

Similarly, maximum increase (38.44 %) in plant dry biomass of Super cash (Table 1) under the foliar application of melatonin was recorded (186.16 g) as compared to without application of melatonin i.e. control (134.47 g). followed by T60 F1 presented the 37.90 % increase in dry biomass (174.89 g) as compared to control (126.82 g). Similarly, heat-sensitive genotypes of tomato expressed that greater plant dry biomass was observed in Nagina (101.22 g) as compared to control (79.17 g) showing 27.85 % increase in plant drybiomass. Whereas, Naqeeb showed 27.11 % improvement inplant dry biomass (94.38 g) as compared to control (74.25 g).

The results (Table 1) showed that under the foliar application of melatonin, 45.65 % increased leaf area was observed in T60 F1 (250.86 cm²) as compared to without application of melatonin i.e. control (172.24 cm²). Whereas Super cash F1 showed 42.33 % increase in leaf area (237.12 cm²) when compared with the control (166.59 cm²). In the case of heat-sensitive genotypes of tomato, the results revealed a greater leaf area in Nagina (36.56 %) and Naqeeb (35.72 %) (heat sensitive) cultivars than their respective control.

The results revealed that the maximum (45.16 %) increase in stem girth (Table 1) under the foliar application of melatonin was noted in Super cash F1 (2.25 cm) as compared to without application of melatonin i.e. control (1.55 cm). Whereas T60 F1 under foliar application of melatonin presented 42 % increase in stem girth (2.13 cm) as compared to the control (1.50 cm). Similar to heat-tolerant genotypes of tomato, foliar application of melatonin also increased the stem girth in heat-sensitive genotypes of tomato and results expressed 35.60 % increase in stem girth in Nagina (1.77 cm) as compared to control (1.32 cm). Whereas, Naqeeb under foliar application of melatonin showed 30.43 % impeovement in stem girth (1.50 cm) as compared to control (1.15 cm).

3.2 Impact of melatonin foliar treatment on quality attributes of tomato genotypes

The greater production of protein (Table 1) from heat-tolerant genotypes of tomato was observed in T60 F1 (65.55 µg g⁻¹ FW) as compared to without application of melatonin i.e. control (48.84 µg g⁻¹ FW) showing 34.21 % increase over control treatment. Whereas Super cash F1 under foliar application of melatonin showed the 32.66 % increase in protein contents (62.30 µg g⁻¹ FW) as compared to control (46.96 µg g⁻¹ FW). In the case of heat-sensitive genotypes of tomato, the 26.52 % increase inprotein contents were recorded in Naqeeb (52.16 µg g⁻¹ FW) as compared to control (41.32 µg g⁻¹ FW). While Nagina under foliar application of melatonin presented only 23.84 % improvement in protein contents (50.01 µg g⁻¹ FW) as compared to its control (40.38 µg g⁻¹ FW). The foliar treatment of melatonin significantly (p ≤ 0.05) increased the protein contents of all growing cultivars either heat tolerant or heat sensitive.

The foliar treatment of melatonin significantly (p ≤ 0.05) increased (Table 1) the number of viable seeds per fruit in all cultivrs of tomato (Table 1). The similar trend of results was observed in the case of the number of viable seeds per fruit of tomato. The maximum increase in (6.07 %) number of viable seeds per fruit from heat-tolerant genotypes of tomato was observed in Super cash F1 (95.64) as compared to without the application of melatonin (90.16). Whereas T60 F1 under foliar application of melatonin showed 6.02 % increase in number of viable seeds per fruit (93.90) as compared to control (88.28).

Similarly in the case of heat-sensitive genotypes of tomato a significant increase (3.72 %) in number of viable seeds per fruit was observed in Naqeeb (83.78) as compared to the control (80.77). While Nagina under foliar application of melatonin presented the 4.49 % more number of viable seeds per fruit (81.45) as compared to the control (77.95).

3.3 Impact of melatonin foliar treatment on physiological traits of tomato genotypes

Data regarding photosynthetic rate (Fig. 1) reflected that 42.04 % increase in photosynthesis rate from heat tolerant genotypes of tomato was observed in Super cash F1 (28.14 μmol CO₂ m⁻² S^-1) as compared to without application of melatonin i.e. control (19.81 μmol CO₂ m⁻² S^-1). However, T60 F1 under foliar application of melatonin showed 39.95 % increase in photosynthesis rate (26.38 μmol CO₂ m⁻² S^-1) as compared to control (18.85 μmol CO₂ m⁻² S^-1).

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Fig. 1

Foliar application of melatonin improved Physiological [Photosynthetic rate (a), Transpiration rate (b), Stomatal conductance (c), SPAD Chlorophyll contents (d)] in heat tolerant and heat sensitive genotypes of tomato under heat stress. Mean value ± standard error, significant difference is exhibited by lower case letters (p ≤ 0.05) according to LSD.

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In case of heat-sensitive genotypes of tomatoes, the greater photosynthesis rate was observed in Naqeeb (21.64 μmol CO₂ m⁻² S^-1) as compared to control (16.26 μmol CO₂ m⁻² S^-1) indicating 33.08 % improvement over control. Whereas, Nagina under foliar application of melatonin presented 31.73 % improvement in photosynthesis rate (18.39 μmol CO₂ m⁻² S^-1) as compared to its respective control (13.96 μmol CO₂ m⁻² S^-1). The foliar treatment of melatonin, statistically (p ≤ 0.05) increased the photosynthesis rate in heat tolerant as well as in heat sensitive cultiars of tomato.

In the same way, significant (p ≤ 0.05) results of melatonin foliar spray swere obtained in case of transpiration rate (Fig. 1). According to the results, maximum transpiration rate was noted in Super cash F1 (2.31 mmol H₂O m⁻² S^-1) as compared to control (1.78 mmol H₂O m⁻² S^-1) reflecting 29.77 % increase over control. Similarly, T60 presented 27.93 % more transpiration rate (2.29 mmol H₂O m⁻² S^-1) as compared to its respective control (1.79 mmol H₂O m⁻² S^-1). Similarly, in heat-sensitive genotypes of tomato, results expressed that the 23.27 % increase in transpiration rate was observed in Naqeeb (1.96 mmol H₂O m⁻² S^-1) as compared to control (1.59 mmol H₂O m⁻² S^-1). While Nagina reflected minimum increase (23.53 %) in transpiration rate (1.89 mmol H₂O m⁻² S^-1) than its control (1.53 mmol H₂O m⁻² S^-1).

The results (Fig. 1) indicated that under the foliar application of melatonin improved 26.63 % stomatal conductance significantly (p ≤ 0.05) in T60 F1 (2.33 mmol m⁻² S^-1) than its control (1.84 mmol m⁻² S^-1). While Super cash F1 genotype showed 27.93 % improvement in stomatal conductance (2.29 mmol m⁻² S^-1) as compared to its respective control (1.79 mmol m⁻² S^-1). Similarly, heat-sensitive genotypes of tomatoes also revealed the same trend as heat-tolerant genotypes.

It is clear from the data (Fig. 1) regarding chlorophyll contents that foliar application of melatonin increased chlorophyll contents significantly (p ≤ 0.05) in all tomato cultivars. The results showed that under the foliar application of melatonin greater chlorophyll contents from heat-tolerant genotypes of tomato were observed in T60 F1 (29.04 mg g⁻¹) as compared to control (21.23 mg g⁻¹) untreated showing 36.78 % increase in chlorophyll contents. Super cash F1 under foliar application of melatonin indicated 39 % improvement in chlorophyll contents (28.12 mg g⁻¹) as compared to its respective control (20.23 mg g⁻¹).

In the case of heat-sensitive genotypes of tomato, the results revealed that foliar application of melatonin increase 26.95 % chlorophyll contents in Naqeeb (21.86 mg g⁻¹) as compared to control (17.22 mg g⁻¹). Whereas, Nagina presented 25.16 % more chlorophyll contents (21.19 mg g⁻¹) as compared to control (16.93 mg g⁻¹).

The results (Fig. 2) regarding WUE had revealed that the highest increase in WUE (9.43 %) was noted in the Super cash F1 (12.18 μmol CO₂/ mmol H₂O) genotype as compared to the control (11.13 μmol CO₂/ mmol H₂O). In the same way, T60 F1 under foliar application of melatonin presented 9.41 % increase in WUE (11.51 μmol CO₂/ mmol H₂O) as compared to its control (10.52 μmol CO₂/ mmol H₂O). Similar to heat-tolerant genotypes, 8.03 % improvement in WUE in heat-sensitive genotypes of tomato was observed in Naqeeb (11.03 μmol CO₂/ mmol H₂O) as compared to control (10.21 μmol CO₂/ mmol H₂O). Subsequently, Nagina under foliar application of melatonin showed greater WUE (9.71 μmol CO₂/ mmol H₂O) than its respective control (9.10 μmol CO₂/ mmol H₂O) indicating 6.70 % increase over control.

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Fig. 2

Foliar application of melatonin improved Water use efficiency (WUE) (a), Leaf temperature (b), and reduced Electrolyte leakage (%) (c), Hydrogen per oxide (H₂O₂) (d) in heat tolerant and heat sensitive genotypes of tomato under heat stress. Mean value ± standard error, significant difference is exhibited by lower case letters (p ≤ 0.05) according to LSD.

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The leaf temperature (Fig. 2) of heat-tolerant genotype T60 F1 (24.20 °C) showed 4.53 % low leaf temperature as compared to control (25.35 °C). Leaf temperature of other heat tolerant genotypes Super cash F1 was also reduced (25 °C) as 5.12 % over its control (26.35 °C). On the other hand, 2.20 % lower leaf temperature in heat-sensitive genotypes of tomato in Nagina (26.60 °C) was observed as compared to control (27.20 °C). Whereas, another heat-sensitive cultivar Naqeeb also showed 2.98 % lower leaf temperature (26 °C) than its respective control (26.80 °C).

The results regarding electrolyte leakage (EL) indicated that the minimum EL was recorded in Super cash F1 (12.15 %) as compared to control (17.76 %). Similarly, T60 F1 under foliar application of melatonin presented the lowest electrolyte leakage (13.24 %) as compared to control (18.23 %). Similar to heat-tolerant genotypes of tomato foliar application of melatonin also decreased the electrolyte leakage in heat-sensitive genotypes of tomato and results expressed that the lowest electrolyte leakage was observed in Nagina (18.67 %) than its control (22.83 %). Whereas, Naqeeb also showed minimum electrolyte leakage (19.15 %) than its control (23.83 %).

3.4 Impact of melatonin foliar treatment on ROS of tomato genotypes

The maximum reduction (13.15 %) in H₂O₂ contents (Fig. 2) under the foliar application of melatonin from heat tolerant genotypes of tomato was noted in T60 F1 (8.24 µmol g⁻¹ FW) as compared to without application of melatonin (13.15 µmol g⁻¹ FW). Similarly, in another heat tolerant cultivar Super cash F1 also presented 39.50 % reduction in H₂O₂ contents (7.52 µmol g⁻¹ FW) than its respective control (12.43 µmol g⁻¹ FW). In case of heat sensitive genotypes of tomato,24.72 % reduction in H₂O₂ contents were observed in Naqeeb (13.43 µmol g⁻¹ FW) when compared with control (17.84 µmol g⁻¹ FW) and followed by Nagina showing the 26.61 % less reduction in H₂O₂ (11.72 μmol g⁻¹ FW) as compared to its control (15.97 µmol g⁻¹ FW).

3.5 Impact of foliar melatonin treatment on leaf and root osmoprotectants (GB and proline) of tomato genotypes

The maximum increase in GB contents (Fig. 3) in leaf and roots of heat-tolerant genotypes of tomato was noted in T60 F1 (22.76 % and 17.14 %) in leaf and roots respectively as compared to without application of melatonin. Similarly, in another heat-tolerant cultivar Super cash F1 also presented 20 % and 15.53 % higher GB contents in leaf and roots than its respective control. In the case of heat-sensitive genotypes of tomato, 13.33 % and 9.52 % more GB contents in leaf and roots were observed in Naqeeb as compared to the control followed by Nagina (12.78 % and 9.30 %) showed high GB contents in leaf and roots respectively as compared to their respective control.

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Fig. 3

Foliar application of melatonin improved osmoprotectants [Leaf GB (a), Root GB (b), Leaf proline (c), Root proline (d)] in heat tolerant and heat sensitive genotypes of tomato under heat stress. Mean value ± standard error, significant difference is exhibited by lower case letters (p ≤ 0.05) according to LSD.

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The maximum increase in proline contents (Fig. 3) in leaf and roots of heat tolerant genotypes of tomato was noted in T60 F1 (20.95 % and 15.05 %) respectively as compared to without application of melatonin. Similarly, in other heat-tolerant cultivar Super cash F1 also presented 20.88 % and 14.28 % higher proline contents in leaf and roots respectively than their respective control. In case of heat-sensitive genotypes of tomato, maximum increase in proline contents of leaf (13.28 %) and roots (10.81 %) were observed in Naqeeb as compared to the control followed by Nagina showed 13.70 % and 11.26 % increase in proline contents in leaf and roots respectively as compared to their respective control.

3.6 Impact of melatonin foliar treatment on enzymatic antioxidant activities of tomato genotypes

The enzymatic antioxidants activities (NOS, NR, GR, and GST) in all genotypes (heat tolerant and heat sensitive) of tomato were significantly (p ≤ 0.05) improved (Fig. 4) when melatonin was applied as compared to control which remained under heat stress without melatonin treatment. Maximum increase in activity of NOS (133.91 %), NR (48.38 %), GR (59.76 %) and GST (55.92 %) was found in heat tolerant genotype (Super cash F1) under heat stress along with melatonin foliar spray. However, both heat tolerant cultivars of tomato showed a non-significant difference in increasing activity of enzymes. while minimum activity of enzymes were indicated by heat sensitive genotypes (Naqeeb and Nagina) as compared to control treatment which remained untreated bearing heat stress.

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Fig. 4

Foliar application of melatonin improved Nitric Oxide Synthase (NOS) activity (a), Nitrate reductase (NR) activity(b), glutathione reductase (GR) activity(c), glutathione S-transferase (GST) activity (d) in heat tolerant and heat sensitive genotypes of tomato under heat stress. Mean value ± standard error, significant difference is exhibited by lower case letters (p ≤ 0.05) according to LSD.

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3.7 Pearson association between morphological, physiological, quality and enzymatic traits of tomato genotypes

The Pearson correlation analysis revealed that there was a significant relationship between morphological, physiological, quality and enzymatic traits of tomato genotypes (Fig. 5). Moreover, the photosynthetic rate, transpiration rate, stomatal contents, chlorophyll contents, WUE, GB, proline, NOS, NR, GR, GST, plant fresh biomass, plant dry biomass, leaf area, stem girth, protein contents, and number of viable seeds were positively associated with each other (p ≤ 0.05) and negatively associated with the EL, leaf temperature, and H₂O₂.

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Fig. 5

Correlation analysis of Photosynthetic rate (PR), Transpiration rate (TR), Stomatal conductance (SC), SPAD chlorophyll contents (Chl), Water use efficiency (WUE), Leaf temperature (LT), reduced Electrolyte leakage (EL), Hydrogen per oxide (HO), Leaf GB, Root GB, Leaf proline (LP), Root proline (RP), Nitric oxide synthase (NOS) activity, Nitrate reductase (NR) activity, Glutathione reductase (GR) activity, Glutathione S-transferase (GST) activity, Plant fresh biomass (PFB), Plant dry biomass(PDM), Leaf area (LA), Stem grith (SG), Protein contents (PC), Viable seeds (VS) in heat tolerant and heat sensitive genotypes of tomato under heat stress.

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