Advertisement

Enhancing thermotolerance of tomato plants (Lycopersieon esculentum Mill.) by heat hardening of seeds

  • Sohair K. IbrahimEmail author
  • Lulwa A. El- Muqadam
Open Access
Research
  • 78 Downloads

Abstract

Background

High temperature is a crucial problem in growing good crops of high temperature sensitive vegetables including tomato. Therefore, this study was carried out to investigate the effects of pre-sowing heat treatments of tomato seeds on germination, growth and biochemical changes of the plants grown under high temperature stress.

Material and methods

The study included two experiments; experiment I dealt with the effect of pre-sowing heat hardening of tomato (Lycopersiecon esculentum Mill. cv. Marmand VF) for different periods of soaking at 25 °C for 5 min, 3 h and 6 h before exposing to 50 °C, 60 °C and 70°C for 0.5 h, 1 h and 2 h.

Results

Best results represented by germination parameters were obtained by soaking the seeds for 5 min before exposure to 50 °C, 60 °C and 70 °C for 0.5 h, 1 h and 2 h. According to the germination data, these treatments were chosen to study their effect on vegetative growth as well as some biochemical parameters (experiment II). The study showed that seed hardening increased growth criteria expressed by stem length, number of branches, number and area of leaves and fresh and dry weight of shoots. The same treatments increased photosynthetic pigments, i.e. (chlorophylls “a” and “b”) and carotenoids of tomato leaves as well as the studied chemical constituents of shoots (reducing sugars, sucrose, amino acids, proline and proteins as well as nucleic acids and saturated fatty acids. Maximum response was attained by treatment with 60 °C for 1 h and 2 h.

Conclusion

Thus, these treatments can help the plant to cope with the adverse effects of high temperature prevailing during their growth stage.

Keywords

Tomato Seed hardening High temperature stress Growth Metabolism 

Introduction

Tomato (Lycopersicon esculentum Mill.) is one of the widely grown vegetable crops. It is an important crop for human consumption.

It is usually cultivated in the open field in Saudi Arabia during September as the prevailing temperature is suitable for growth and development of the plant. Tomato plants grow rapidly, so extending the season and increasing continuity of supply can be achieved by sowing the seeds during spring. However, the plants exhibited to high temperature during their growth and development.

High temperature is a crucial problem in growing good crops of high temperature sensitive vegetables including tomato. It impairs different morphological criteria (Khalil and Moursy 1983 and Warrag 1999). High temperature affects wide spectrum of both biochemical and physiological responses within the plant cell. These results are expected and described by many researchers especially in the case of growing organs, since all the reactions in the plant already take place rapidly and further rise in temperature might easily disturb the balance (Fisher 1980). Other investigators reported that extreme and variation of high temperature can damage the intermolecular interactions needed for growth (Bita and Gerats 2013).

Protection of plants from high temperature stress can be achieved by several mechanisms; one of these mechanisms is seed hardening; it is a physiological seed enhancement method (Taylor et al. 1998). Seed hardening can be achieved by pre-sowing treatments in which the seeds are soaked in water, an osmotic solution or growth regulators as well as exposing the seeds to elevated temperature above the maximum temperature prevailing during their growth. These treatments allow the seeds to go to the first stages of germination but not permit radicale protrusion through the seed coat (Heydeker 1977). Planting hardened seeds in the field gives the plant a better start than the non-hardened plants. Thus, hardened plants might survive adverse environmental stresses like high temperature more easily because of the advanced state of development.

The present study is an additional contribution for understanding the effects of pre-sowing heat treatments of tomato seeds on germination, growth and biochemical changes of the plant grown under high temperature stress (30–40°C).

Material and methods

The study comprised of two experiments; the planning of the second experiment depended on the results of the first one according to the following order:

Experiment I: germination tests

Uniform seeds of tomato (Lycopersicon esculentum Mill. cv. Marmand VF) were soaked in water at 25 °C for 5 min, 3 h and 6 h and dried quickly within two layers of filter paper, and each group was divided to two sets. The first set from each group was weighed then dried at 105 °C till constant weight according to Hart and Neustadt (1957) to measure moisture content that reached 20.6%, 44.33% and 63.52%, respectively. The second set from each group was subjected to 50, 60 and 70 °C for 0.5 h, 1 h and 2 h. The seeds were germinated at 25 °C in Petri dishes each contained 20 seeds.

Ten replicates were allotted for each treatment as well as control treatment (seeds soaked for 0.5 h, 1 h or 3 h at room temperature (25 °C). Germination was recorded for 8 days, and then germination data were recorded as follows: germination capacity, radical and hypocotyl length as well as radical fresh weight and seedling fresh weight.

Experiment II

The previous experiment results indicated that the best results were obtained and the seeds were then soaked for 5 min (moisture content 20.6%) and exposed to for 50 °C, 60 °C and 70 °C for 0.5 h, 1 h and 2 h as well as seeds soaked for 5 min only as (control). These treatments were chosen to study their effect on vegetative growth and some biochemical parameters.

Growing technique and sampling

Tomato seeds were sown on March in JV (7) cubes, and when the seeds were attained 30 days, they were transferred to 30 cm in diameter pots filled with equal amounts of soil (consisted of clay + paet moss + perlite at the ratio of 1:3:5 (w/w). Fertilization was applied as the recommended dose (5 g superphosphate, 10 g mixture of ammonium sulphate and potassium sulphate at the ratio of 3:2) for each pot.

The plants were supplied with water according to their requirements which was governed by climatic conditions. The experiment was carried outdoors in the screen of the Girls College of Science, Damman, Saudi Arabia, for two successive seasons. The maximum and minimum temperatures as well as relative humidity are shown in Table 1.
Table 1

Average of monthly maximum and minimum air temperature and relative humidity during the two seasons of study

Month

1st season

2nd season

Temperature

Relative humidity %

Temperature

Relative humidity %

Maximum

Minimum

Maximum

Minimum

Maximum

Minimum

Maximum

Minimum

March

30

10

95

17

30

11

95

20

April

38

14

85

10

36

14

95

17

May

43

16

98

3

44

18

90

8

Samples were collected at random 30 days after transplanting (DAT). Each treatment was divided into three replicates for recording vegetative characters as well as chemical analysis.

Biochemical analysis

Photosynthetic pigments [chlorophyll a, chlorophyll b and carotenoids (car)] were determined in fresh leaves (Metzner et al. 1965).

The following parameters were estimated in dry shoots at 70 °C

Reducing sugars, sucrose and polysaccharides were measured according to (Dubios et al. 1956). Total amino acids were analysed according to Boulter and Barber (1963). Protein extraction followed Anderson and Beardall (1991) and estimated as described by Lowry et al. (1951).

Proline, nucleic acids and fatty acids were determined in fresh shoots. Proline was measured according to Troll and Lidsley (1955). Nucleic acids were extracted according to Schmidt and Thaunhauser (1945), and ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were estimated according to the method of Winzler (1955) and Burton (1956), respectively.

Fatty acids were analysed through four successive steps: (1) extraction by petroleum ether 60–40 °C, (2) saponification with NaOH (20%), (3) methylation by methyl alcohol and (4) identification by GLC (Varian Model 6000 chromatography). The GLC condition was as follows: The glass column filled with 15% DEGS. The column oven temperature was programmed at 6 °C/min from 80 °C to 130 °C and kept finally for 25 min. Injector and detector temperatures were 220 °C and 260 °C, respectively. Gases’ flow rates were 30, 30 and 300 cm/min for N2, H2 and air, respectively. The flow rate inside the column was adjusted as 1 ml/min.

Statistical analysis

The data of the first experiment was subjected to statistical analysis according to the analysis of variance with interaction. The values of the least significant difference (LSD) were calculated at 5% level of probability.

The data of the second experiment was arranged in complete randomized design. The obtained data were statistically analysed according to Duncan (1955) at the probability of 5%. Mean value followed by the same letter within each column is not significant.

By applying the Steel and Torrie (1966) test, the results showed the same trend. Therefore, the combined analysis of the two seasons was calculated. The combined analysis of the two seasons was calculated after Steel and Torrie (1966) as the results obtained showed the same trend.

Results

Germination percentage (Table 2) showed the optimum values as the seeds soaked for 5 min, 3 h and 6 h at room temperature compared to the other treatments at the different degrees (50, 60 and 70 °C). Moreover, these treatments decreased germination percentage by increasing the degree of temperature from 50 → 60 → 70 °C as well as prolonging the period of soaking from 5 min → 3 h → 6 h. The lowest value of germination percentage was obtained by soaking the seeds for 3 h or 6 h before exposure for 70 °C for any period of exposure. Thus, the seeds did not tolerate the high percentage of moisture content concomitant to high temperature.
Table 2

Effect of seed hardening on germination percentage, radicale length (cm) and hypocotyl length (cm)

Treatment

Germination %

Radicale length (cm)

Hypocotyl length (cm)

Temp (°C)

Exposure period (h)

Period of soaking

Period of soaking

Period of soaking

5 min

3 h

6 h

5 min

3 h

6 h

5 min

3 h

6 h

Control

99.8

99.5

99.4

4.17

4.37

4.30

11.90

12.00

11.13

50

0.5

99.4

95.4

96.1

5.13

4.80

4.60

13.03

12.40

11.63

1

98.0

92.8

92.7

5.33

5.03

4.50

12.65

11.40

11.17

2

97.8

89.2

80.5

4.67

4.63

4.93

13.03

12.07

11.70

60

0.5

99.6

77.4

74.0

5.23

4.57

4.70

11.80

12.20

10.13

1

97.4

75.2

61.0

5.47

4.23

4.23

12.23

11.80

10.17

2

95.6

68.8

61.5

5.47

4.20

4.17

12.03

11.50

10.13

70

0.5

91.8

55.4

52.2

4.70

4.07

3.53

12.13

10.50

10.13

1

84.9

51.5

50.4

4.63

3.90

3.10

11.70

8.50

8.63

2

85.5

49.9

50.6

4.66

3.20

3.13

10.73

8.13

7.47

LSD of interaction

0.49

0.72

Results in Table 3 revealed the interaction of the soaking period of the seeds and the period of exposure to different degrees of temperature on the radicale length; their length significantly increased by exposing the seeds to 50 °C or 60 °C for all exposure periods after soaking the seeds for 5 min. However, soaking the seeds for 6 h before exposing to 70 °C at all used periods decreased significantly the length of radicale as compared to control.
Table 3

Effect of seed hardening on radicale fresh weight (g/plant) and seedling fresh weight (g/plant)

Treatment

Radicale fresh wt. (g/plant)

Seedling fresh wt. (g/plant)

Temp (°C)

Exposure period (h)

5 min

3 h

6 h

5 min

3 h

6 h

Control

0.001

0.011

0.012

0.081

0.080

0.080

50

0.5

0.012

0.011

0.012

0.085

0.077

0.077

1

0.012

0.011

0.011

0.087

0.075

0.077

2

0.012

0.011

0.011

0.082

0.072

0.072

60

0.5

0.012

0.010

0.011

0.087

0.073

0.074

1

0.012

0.009

0.010

0.088

0.074

0.072

2

0.012

0.009

0.009

0.083

0.070

0.069

70

0.5

0.011

0.009

0.008

0.084

0.055

0.051

1

0.011

0.008

0.008

0.082

0.056

0.051

2

0.011

0.007

0.007

0.082

0.055

0.050

LSD of interaction

0.001

0.003

Hypocotyl length significantly increased by soaking tomato seeds for 5 min before exposing to the different used periods at 50 °C. On the other hand, increasing the period of soaking to 3 h and 6 h accompanied with elevating the exposure temperature to 60 °C or 70 °C significantly decreased hypocotyl length. Maximum significant decrease was reached by soaking the seeds for 3 h or 6 h before exposing to different periods at 70 °C (Table 2).

Radicale fresh weight did not show any significant increase due to treatments. In addition, significant decrease was obtained by soaking the seeds for 3 h or 6 h before exposing to 60 °C or 70 °C for all exposure periods (Table 3).

Seedling fresh weight exhibited significant increase by soaking the seeds for 5 min before exposing to 50 °C or 60 °C for 0.5 h or 1 h. However, prolonging the period of soaking to 3 h and 6 h before exposing the seeds to different temperature degrees for all used periods of exposure (0.5 h, 1 h and 2 h) showed an opposite trend as all treatments decreased the fresh weight of the seedling (Table 3).

Growth responses

The studied parameters of growth were shown in Table 4. Stem length of tomato plants showed an increase by exposing the seed for 60 °C or 70 °C. Significant increments were attained by treatment of 60 °C for 2 h or 70 °C for 0.5 h.
Table 4

Effect of seed hardening on vegetative growth

Treatments

Parameters

Temp. (°C)

Exposure period (h)

Stem length (cm)

No. of branches/plant

No. of leaves/plant

Leaf area (cm2/plant)

Leaves fresh wt. (g/plant)

Leaves dry wt. (g/plant)

Shoot Fresh wt. (g/plant)

Shoot dry wt. (g/plant)

Control

36.5b

0.67d

6.33c

199.27e

6.01g

0.691d

11.76e

1.41ef

50

0.5

35.1b

0.67d

6.67c

229.10bc

6.51f

0.714cd

12.52d

1.44de

1

36.0b

1.00c

7.67ab

238.70b

6.76ef

0.743bc

13.20c

1.48cd

2

37.7b

1.33b

8.33b

239.70b

7.18cd

0.779b

13.53c

1.51bc

60

0.5

36.5b

1.00c

7.67c

215.90cd

7.02de

0.764b

13.69c

1.49cd

1

36.9ab

1.00c

8.00c

287.40a

7.65ab

0.764b

14.79b

1.51bc

2

39.3a

1.67a

8.33a

279.00a

7.95a

0.829a

15.77a

1.63a

70

0.5

39.0a

1.33b

7.67b

252.0b

7.46bc

0.774b

14.78b

1.54b

1

37.4ab

1.00c

7.00c

220.00cd

7.73ab

0.723c

13.74c

1.47cd

2

37.1ab

1.00c

6.67c

208.30de

6.10g

0.683d

12.05de

1.36f

LSD at 5%

2.40

0.25

0.88

15.31

0.883

0.031

0.53

0.05

The number of branches significantly increased by all treatment seeds at 50 °C for 0.5 h, and the highest number of branches obtained by treatment at 60 °C as well as treatment at 70 °C for 0.5 h. Maximum significant increase attained by exposing the seeds to 60 °C for 1 h or 2 h.

The number of leaves significantly increased by treatment at 50 °C for 1 h or 2 h and 60 °C for all periods of exposure.

The area and fresh weight of leaves increased significantly by all treatments with the exception of seeds by treatment at 70 °C for 2 h. Maximum increase was attained by treating the seeds for 2 h at 60 °C. The dry weight of leaves followed almost the same trend but the increase with the treatment at 50 °C for 0.5 was not significant (Table 4).

Shoot fresh weight showed a significant increase with all treatments except treating the seeds at 70 °C for 2 h.

Shoot dry weight followed the same pattern of changes shown in the case of fresh weight. Meanwhile, the increase with treatment at 50 °C for 0.5 h was not significant (Table 4).

Biochemical analyses

Results recorded in Table 5 show the effect of hardening on photosynthetic pigments and the studied carbohydrate fractions. Chlorophylls a significantly increased with all treatments except those exposed to 50 °C for 0.5 h. The maximum increase was given by exposing the seeds to 60 °C for 2 h followed by treatments at 60 °C or 70 °C for 1 h. Chlorophylls b content significantly increased with treatment at 60 °C for 2 h and 70 °C for all used exposure periods. Total chlorophylls (a + b) showed a significant increase by all hardening treatments except 50 °C or 60 °C for 0.5 h.
Table 5

Effect of seed hardening on photosynthetic pigments (mg/gm fresh weight of leaves) and carbohydrate content of shoots (mg/glucose/g dry wt.)

Treatments

Parameters

Temp. (°C)

Exposure period (h)

Ch1a

Ch1b

Ch1 a + b

Carotenoids

Reducing sugars

Sucrose

Polysaccharides

Control

0.563e

0.225d

0.788fg

0.217f

18.26e

32.11d

137.90a

50

0.5

0.565e

0.210e

0.785g

0.222ef

18.01e

30.92d

140.73a

1

0.580d

0.232cd

0.812e

0.231de

24.43b

36.35b

125.62c

2

0.603c

0.230cd

0.833d

0.238d

22.37cd

34.55c

112.06d

60

0.5

0.581d

0.226d

0.807ef

0.239d

22.37cd

38.07b

114.14d

1

0.642b

0.230cd

0.872bc

0.299a

27.14a

46.14a

89.96f

2

0.683a

0.250a

0.933a

0.308a

28.22a

44.92a

103.15e

70

0.5

0.611c

0.248ab

0.883b

0.285b

22.55c

38.22b

112.36d

1

0.642b

0.241bc

0.859c

0.268c

21.06d

34.57c

113.20d

2

0.617c

0.238bc

0.855c

0.262c

22.14cd

31.91d

131.09b

LSD at 5%

0.014

0.011

0.020

0.009

1.32

1.76

5.07

Carotenoid content showed a significant increase with all treatments except 50 °C for 0.5 h. The highest value was obtained by exposing the seeds to 60 °C for 1 h or 2 h.

Carbohydrate fractions showed that direct reducing sugars were significantly increased for all treatments except the treatment at 50 °C for 0.5 h, 50 °C for 0.5 h and 60° for 1 h or 2 h.

Sucrose content followed more or less the same trend of reducing sugars with all treatments except the treatment at 70°C for 2 h, and treatment at 60 °C for 1 h or 2 h resulted in the highest significant increase.

Polysaccharide content clearly showed an opposite trend to reducing sugars and sucrose. Significant decrease recorded for all treatments except for treatment at 50 °C for 0.5 h. Maximum decrease was recorded by treatment at 60 °C for 1 h followed by 2 h (Table 5).

Table 6 shows the changes of proline, amino acids and protein as well as nucleic acids RNA and DNA. Proline content exhibited significant increment, with the exception for all treatments except for the treatment at 50 °C for 0.5. Maximum increase was obtained by the treatment at 60 °C for 1 h followed by 2 h.
Table 6

Effect of seed hardening on proline, free amino acids, RNA and DNA content of plant shoots

Treatments

Parameters

Temp. (°C)

Exposure period (h)

Proline, ug/g fresh wt.

Amino acids, mg/g dry wt.

Proteins, mg/g dry wt.

RNA, mg/g fresh wt

DNA, mg/g fresh wt.

Control

41.22g

7.01ef

29.21e

1.01d

0.211c

50

0.5

42.22g

6.61fg

28.38e

0.979d

0.211c

1

44.77f

7.22e

30.00de

1.07c

0.219a

2

47.25e

7.43de

30.95cd

1.10c

0.214bc

60

0.5

49.15c

6.40g

28.55g

1.17b

0.215abc

1

62.14b

8.11bc

32.04c

1.23a

0.218ab

2

64.48a

9.52ab

36.05a

1.19ab

0.218ab

70

0.5

49.33e

7.95cd

33.93b

1.09c

0.217ab

1

55.15c

9.92a

31.45c

1.01d

0.211c

2

52.14d

9.18b

31.52c

0.966d

0.212c

LSD at 5%

2.12

0.56

1.37

0.050

0.004

Amino acids significantly increased with all treatments at 60 °C or 70 °C. The highest value of increase was obtained by 60 °C for 2 h as well as 70 °C for 1 h.

Protein content exhibited significant increments by all treatments with the exception of two treatments, 50 °C or 60 °C for 0.5 h. The highest value of significance was obtained by the treatment at 60 °C for 1 h followed by the treatment for 2 h.

Nucleic acid (RNA) recorded a significant increase for most treatments except 50 °C for 0.5 h as well as 70 °C for 1 h or 2 h. Meanwhile, maximum significant increase was recorded by treatment at 60 °C for 1 h or 2 h. DNA content showed a significant increase by the following treatments: 50 °C for 1 h, 60 °C for 1 h or 2 h and 70 °C for 1 h (Table 6).

Table 7 shows the effect of heat hardening of tomato seeds on the percentage of fatty acids. The analysis shows an increase of myristic acid percentage, while it shows a decrease of palmitic oleic and palmitoleic percentage. Total saturated fatty acid percentage was increased obviously compared to untreated plants. Maximum increments were recorded by treatment at 60 °C for 2 h.
Table 7

Effect of seed hardening on the percentage of fatty acids in plant shoots

Treatment

Percentage of saturated and unsaturated fatty acidsa

Percentage of total saturated fatty acids

Temp. °C

Exposure period (hr)

C12: 0

C14:0

C14:1

C16:0

C16:1

C16:2

C18:0

C18: 1

C18:2

C18:3

Control

0.69

1.32

36.61

31.61

1.45

7.26

16.88

1.86

45.88

 50

2

0.29

58.70

12.85

4.99

0.93

0.97

13.82

3.36

72.81

 60

1

2.81

50.64

5.07

7.00

11.45

0.64

10.26

1.51

2.38

70.71

 60

2

0.07

67.43

3.14

8.21

4.47

1.23

3.89

3.98

1.82

1.10

79.60

 70

0.5

0.30

57.94

11.31

11.5

0.32

0.34

10.60

3.10

1.58

69.88

aC12:0 lauric acid; C14: 0 myristic acid; C14: 1 nyristoleic acid; C16:0 palmitic acid; C16:1 palmitoleic acid C16: 2 palmitolenic acid; C18: 0 stearic acid; C18: 1 oleic acid; C18: 2 linoleic acid; C18:3 linolenic acid

Discussion

High temperature is considered one of the most important environmental factors that affect plant growth. It is the most influential factor which induces an increase of plant evaporation demand and indirectly contributes to water deficiency or salt stress (Karim et al. 1998). Seed hardening modulates the physiological and biochemical nature of seeds that lead to induction of the ability of seeds to stand higher temperature for a prolonged period (Sujatha et al. 2013).

It is clear that exposure of seeds to suitable high temperature and period of soaking improved radicale and plumule length as well as seedling fresh weight. However, the increase of temperature and period of exposure caused a harmful effect (Tables 2 and 3). These results coincide with the findings of other investigators (Farooq et al. 2004, 2005; Rehman et al. 2014).

Other researchers stated that seed hardening can modify physiological and biochemical characters that enable seeds to tolerate environmental stress and stand more easily under unsuitable conditions (Matsushima and Sakagami 2013). Increase of radicale and plumule length as well as fresh weight of seedlings indicated many alterations, such as changes within the cytoplasm as hydration of colloids and increase the viscosity and elasticity (Sujatha et al. 2013). Metabolic activity was also suggested by other investigators as soaking of seeds enhancing metabolites (Barsa et al. 2005) and inducing carbohydrate to become ready to be used for cell elongation (Farooq et al. 2006, on rice). All these changes lead to a better start and uniform of the seedling that can endure environmental stress (Farahani et al. 2011). Thus, seed hardening stimulates pregermination metabolic process without protrusion of the radicale through the seed coat (Heydeker 1977) and provides a faster and synchronized germination (Nawaz et al. 2009).

Improvement of vegetative growth represented by enhancement of branching and increase of number and area of leaves as well as fresh and dry weight of tomato leaves and shoots indicates a generally positive effect. Pre-sowing heat hardening of tomato seeds with 50 °C and 60° for 1 h or 2 h showed the highest increments (Table 4). These results may be attributed to healthy germination of seed, which in turn gave the plant a better start and induced further growth of tomato seedlings. These results were supported by the findings of other researchers (Khalil and Moursy 1983, Gamal El-Din 1999 and El-Moursi et al. 2012) who proved that heat hardening of seeds promoted the growth of different plants. In addition, Souza and Devaraj (2013) reported an accumulation of biomass in heat-acclimated Dilchos libalab under heat stress condition.

Photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) were increased by the hardening of tomato seeds. Maximum level was attained by seeds treatment with 60 °C for 2 h (Table 5). Lower level of the photosynthetic pigments in control plants reflects the effect of high temperature stress as the ambient temperature above the threshold (43–44). The impairment of chlorophyll accumulation is the first process occurring in the plastids due to high temperature. Anjum et al. (2011) reported that the decrease of chlorophylls under drought stress may be a result of pigment photo-oxidation and chlorophyll degradation. Other researchers attributed a decrease of chlorophylls to the reduction of their synthesis or acceleration of degradation or combination of both. In support to these finding, Dutta et al. (2009) and Reda and Mandura (2011) showed destruction of numerous enzymes involved in the mechanism of chlorophyll synthesis under high temperature stress. High chlorophyll content due to hardening in the present study improved protection of tomato plants from heat stress as susceptible genotypes showed a higher reduction of total chlorophylls than the tolerant ones (Gosavi et al. 2014 and Zhou et al. 2017). Moreover, other researchers stated that the accumulation of chlorophylls has been used to characterize the variability of thermotolerance for many crop species (Selvaraj et al. 2011).

The increase of carotenoids accompanied with high level of chlorophylls in treated plants pointed clearly to the effective role of carotenoids in protecting chlorophylls from the damage of singlet oxygen. Carotenoids scavenge them through directly quenching the excited triplet state of chlorophyll molecule and dissipate as a heat (Pallet and Young 1993). Kuczyriska et al. (2012) reported that xanthophylls play a key role in minimizing the overoxidation in higher plants. Thus, tomato seed hardening increased photosynthetic pigments (ch1s and carotenoids of tomato plant), which in turn minimize the damage of light-absorbing efficiency of photosystems (PSI and PSII) (Murkowski 2001; Langium et al. 2006 and Souza et al. 2004).

Plants use different strategies to maintain osmotic balance as the synthesis and accumulation of soluble sugars, amino acids and proline (Shao et al. 2007 and Hayat et al. 2012).

Reducing sugars as well as sucrose increased by hardening treatments in the present study especially by 60 °C for 1 h or 2 h treatments. On the other hand, the same treatments showed a pronounced decrease of polysaccharide content (Table 5). Other researchers studied the correlation between soluble sugars and polysaccharides in tolerant and sensitive varieties of plants as the tolerant varieties contain high level of soluble sugars especially sucrose concomitant to high activity of sucrose–phosphate synthetase compared to sensitive ones (Kerr et al. 1987 and Basu et al. 1991). Previously, Dinar and Rudich (1985) showed an increase of sucrose accompanied by a decrease of starch in a tolerant variety of tomato (Robbin) compared to the sensitive one (Roma). Later, other investigators proved that sucrose has a crucial role in increasing the osmotic potential of stressed cells (Ruan et al. 2010). In addition, Greer and Weston (2010) reported an accumulation of total soluble sugars in heat-acclimated varieties of Vitis venefera.

Proline is one of the most important amino acids which were accumulated under stress. The present investigation showed that proline content increased by most of the hardening treatments. Maximum increment was given by 60 °C for 1 h followed by exposure for 2 h. Several studies cleared a good relation between proline and increasing tolerance of plants under environmental stresses. These amino acids have different crucial roles act as hydroxyl scavenger, stabilization of membranes and protein structure, as sink for carbon and nitrogen for stress recovery and buffering cellular redox potential under stress (Hayat et al. 2012; Kavikishor and Sreenivasulu 2014 and Yaish 2015). Moreover, Li et al. (2013) reported inducing of tolerance of maize plants under high temperature stress concomitant to accumulation of proline through P5C5 (Δ" pyrrolidine-5-carbolate synthesis (2.7.2.11)) using hydrogen sulphide. Other researchers reported that an accumulation of proline under high temperature stress allows the plants to cope with heat stress (Chakraborty and Tongden 2005 and Rasheed et al. 2011).

It is worth to mention that heat stress injury involves water deficit and cell turgor as high temperature cause increases in transpiration and in turn these changes lead to water deficit and increments of loss of turgidity (Cansev 2012).

Protection against dehydration due to high temperature stress can occur via osmoprotectant (soluble sugars, amino acids and proline as these metabolites act as stabilizer of cellular membranes and maintain turgor (Farooq et al. 2008). Many researchers proved that tolerant verities of different plants induced osmolytes as soluble sugars and proline under drought and high temperature. (Arunkumar et al. 2012; Han et al. 2013; Devi and Sujatha 2014 and Solanki and Samangi 2014). Thus, accumulation of soluble sugars and proline are one of the potential biochemical indicators in selecting tolerant cultivars and allowing the plant to cope with heat stress.

Total protein showed significant increase due to heat hardening of tomato seeds and treatment at 60 °C for 2 h showed the highest level (Table 6). The same observation was recorded by other researchers (Gulen and Eris 2004 and He et al. 2005). It is worth to mention that tomato plants in the present study were exposed to high temperature during their growth as the ambient temperature reached (30–40°C) (Table 1).

Nucleic acid (DNA and RNA) contents of tomato shoots (Table 6) were increased due to hardening of seeds. Maximum increase was attained by treatment at 60 °C for 1 h or 2 h. The promoting effect of heat hardening overcame the impairment of the prevailing high temperature on tomato plants. Other investigators reported a decrease of DNA and RNA in wheat plants due to high temperature (Sadak and Orabi 2015). Heat stress injury involves water deficit and cell turgor. Other studies showed a decrease of nucleic acid associated with a rise of RNase activity under deficient water supply (Mukherjee and Mukherjee 2015).

High temperature stress induces changes of lipid membranes; it increases their fluidity via decreasing their lipid saturation (Horvath et al. 2012). Thus, it is important to increase the saturation of fatty acids for maintaining stability and enhancement of heat tolerance for membranes (Larkindale and Huang 2004).

The present study showed that heat hardening of seeds increased the percentage of saturated fatty acids treatment at 50 °C for 2 h as well as treatment at 60 °C for 1 h or 2 h increased the ratio of saturated fatty acids to 72.81%, 70.70% and 79.60%, respectively, compared to control.

Therefore, the present data can illuminate that saturation of fatty acids can share in enhancing heat tolerance of tomato plants (Bita and Gerats 2013 and Ibrahim and El-Moqadam 2015).

Conclusion

Finally, it can be concluded that heat hardening of tomato seeds with 60 °C for 1 or 2 h could alleviate the harmful effect of high temperature prevailing during tomato plants’ growth, through the enhancement of their protective parameters such as carotenoids, proline, osmolytes and saturated fatty acids. Thus, this protective mechanism helped the plants to induce their tolerance against high temperature stress, which in turn was reflected on their growth.

Notes

Acknowledgments

The authors are thankful to the National Research Centre for provision of laboratory facilities to carry out this research.

Funding

There are currently no funding sources in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

The datasets generated and/or analysed during the current study are included in this study.

Authors’ contributions

SKI preformed the laboratory analysis and wrote the paper, and LAE performed the data and coordinated the data collection. Both authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Anderson JW, Beardall J (1991) Molecular activities of plant cells: an introduction to plant biochemistry. Blackwell Scientific Publications, London, p 264Google Scholar
  2. Anjum SA, Yie X, Wang L, Saleem MF, Man C, Lei W (2011) Morphological, physiological and biochemical responses of plant to drought stress. Afr J Agric Res 6:2026–2032Google Scholar
  3. Arunkumar R, Sairam RK, Deshmukh PS, Pal M, Sangeeta K, Sunll KP, Kashwaha SR, Singh TP (2012) High temperature stress and accumulation of compatible solution in ckickpea (Cicer arietinum L.). Indian J Plant Physiol 17:145–150Google Scholar
  4. Barsa SMA, Farooq M, Tabassum R, Ahmed N (2005) Seed treatment in fine rice (Oryza sativa L.). Seed Sci Technol 33:623–628CrossRefGoogle Scholar
  5. Basu PS, Minhas JS (1991) Heat tolerance and assimilate transport in different potato genotypes. J Exp Bot 42:860–861CrossRefGoogle Scholar
  6. Bita CE, Gerats T (2013) Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress –tolerant crops. Front Plant Sci 4:1–18CrossRefGoogle Scholar
  7. Boulter D, Barber T (1963) Amino acid metabolism in germinating seed of Vicia faba L. in relation to their biology. New Phytol 62:301–304CrossRefGoogle Scholar
  8. Burton K (1956) A study on the condition for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–317CrossRefGoogle Scholar
  9. Cansev A (2012) Physiological effect of high temperature treatment on leaves of olive cv. Gemlik. Plant Arch 12:521–525Google Scholar
  10. Chakraborty U, Tongden C (2005) Evaluation of heat acctimation and salicylic acid treatmetns as potent inducers of thermotolerance in cicer arientum L. Curr Sci 89:384–389Google Scholar
  11. Devi SPS, Sujatha B (2014) Drought. Induced accumulation of soluble sugars and proline in two pigeon pea (Cajans Cajanus L. Mill sp.) cultivars. Int J Res Dev 3:302–306Google Scholar
  12. Dinar M, Rudich J (1985) Effect of heat stress on assimilate partitioning in tomato. Ann Bot 56:239–248CrossRefGoogle Scholar
  13. Dubios M, Gilles KA, Hamelton JK, Robers PA (1956) Colourimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  14. Duncan DB (1955) Multiple range and multiple F-test. Biometrics 11:1–4MathSciNetCrossRefGoogle Scholar
  15. Dutta A, Mohanty S, Tripathy BC (2009) Role of temperature stress on chloroplast biogenesis and protein important in pea. Plant Physiol 150:1050–1061CrossRefGoogle Scholar
  16. El-Moursi A, Gamal El-Din KA, Tarraf SA (2012) Physiological response of lupine plant (Lupinus temis L.) to heat hardening. Am Eurasian J Agric Environ Sci 12:660–663Google Scholar
  17. Farahani HA, Moaven P, Maroufi K (2011) Effect of thermopriming on germination of cowpea (Vigna seninsis L.). Adv Environ Biol 5:1668–1675Google Scholar
  18. Farooq M, Barsa SMA, Rehman H, Saleem BA (2008) Seed priming enhances the performance of late sown wheat (Triticum aestivum L.) by improving the chilling tolerance. J Agron Crop Sci 194:55–60CrossRefGoogle Scholar
  19. Farooq M, Basra SMA, Ahmed N, Hafeez K (2005) Thermal hardening: a new seed vigor enhancement tool in rice. J Integ Plant Biol 47:187–193CrossRefGoogle Scholar
  20. Farooq M, Basra SMA, Hafeez K, Warriach EA (2004) Influence of high and low temperature treatments on the seed germination and seedling vigor of coarse and fine rice. Int Rice Res Notes 29:69–71Google Scholar
  21. Farooq M, Basra SMA, Khalid M, Tabassum R, Mahmood T (2006) Nutrient homeostasis, metabolism of reserves, and seedling vigor as affected by seed priming in coarse rice. Can J Bot 84:1196–1202CrossRefGoogle Scholar
  22. Fisher RA (1980) Influences of water stress on crop yield in semiarid regions. In: Turner NC, Kramer PJ (eds) Adaptation of plants to water and high temperature stress. Willey Inter. USA, Pub, pp 323–339Google Scholar
  23. Gamal El-Din KM (1999) Vegetative growth, amino acids and alkaloids changes by heat hardening treatments of Hyoscyamus muticus L. Egypt J Appl Sci 14:216–227Google Scholar
  24. Gosavi GU, Jadhav AA, Kale AA, Godakh SR, Pawar BD, Chimoto VP (2014) Effect of heat stress on proline, cholorophyll content, heat shock proteins and antioxidant activity in sorghum (Sorghum bicolor) at seedling stage. Indian J Biotechnol 13:356–363Google Scholar
  25. Greer DH, Weston C (2010) Heat stress affect growth, sugar accumulation and photosynthesis of Vitis vinifera c.v Semillon grapevines grown in controlled environment. Funct Plant Biol 37:206–214CrossRefGoogle Scholar
  26. Gulen H, Eris A (2004) Effect of heat, stress on peroxidase activity and total protein content in strawberry plants. Plant Sci 186:739–744CrossRefGoogle Scholar
  27. Han Y, Fan S, Zang Q, Wang Y (2013) Effect of heat stress ton the MDA, proline and soluble sugars content in leaf lettuce seedlings. Agric Sci 4:112–115Google Scholar
  28. Hart JR, Neustadt MH (1957) Application of the Karl Fisher method to gain moisture determination. Cereal Chem 34:26–30Google Scholar
  29. Hayat S, Hagat Q, Ahmed A (2012) Role of proline under changing environments. Plant Signal Behav 1:1456–1466CrossRefGoogle Scholar
  30. He Y, Lin X, Huang B (2005) Protein changes in acclimated and non-aclimated creeping bentgrass Agostis Palustris, Hads. J Am Soc Hort Sci 130:521–526CrossRefGoogle Scholar
  31. Heydeker W, Joshua A (1977) Alleviation the thermodormancy of lettuce seeds. J Hortic Sci 52:87–98CrossRefGoogle Scholar
  32. Horvath I, Glatz A, Nakamoto H, Mishkind ML, Munnk T, Saidi Y (2012) Heat shock response in photosynthetic organisms: membranes and lipid connections. Prog Lipid Res 51:208–220CrossRefGoogle Scholar
  33. Ibrahim SK, El-Moqadam L (2015) Improving tolerance of tomato plants (Lycopersicon esculentum Mill) by foliar application of benzyl adenine. Middle East J Appl Sci 5:848–854Google Scholar
  34. Karim MA, Fracheboud Y, Stamp P (1998) Heat tolerance of maize with reference to some physiological characteristics. Ann Bangladesh Agric 1:27–35Google Scholar
  35. Kavikishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostatis are more critical tissue. Plant Cell Environ 37:300–311CrossRefGoogle Scholar
  36. Kerr JL, Kimpel J, Nagao RT (1987) Heat shock gene families of soybean and the regulation of their expression. In: Plant gene system and their biology. Alan R. Liss, Inc., Ny, USA, pp 87–97Google Scholar
  37. Khalil S, Moursy HA (1983) Changes in some germination morphological and reproductive characters of tomato plant as influenced by heat treatments of seeds. Ann Agric Sci Fac Agric Ain Shams Univ Cairo Egypt 28:1099–1121Google Scholar
  38. Kuczyriska P, Latowski D, Niczyporuk S (2012) Zeanthin epoxidation-an in vitro approach. Acta Biochem Polonica 59:105–107Google Scholar
  39. Langium CUI, Jianlong LI, Yamin FAN, Sheng XU, Zhen Z (2006) High temperature effects on photosynthesis, PS II functionality and antioxidant activity of two festuca arundinacea cultivars with different heat susceptibility. Bot Stud 47:61–69Google Scholar
  40. Larkindale J, Huang BR (2004) Thermotolerance and antioxidant systems in Agrostis stolonifera involvment of salicylic acid, abscisic acid, calcium hydrogen peroxide and ethylene. J Plant Phystol 161:405–413CrossRefGoogle Scholar
  41. Li ZG, Ding XJ, Du PF (2013) Hydrogen sulphide donor sodium hydro-sulfide improved heat tolerance in maize and involvement of proline. J Plant Physiol 170:741–747ADSCrossRefGoogle Scholar
  42. Lowry OH, Rosebrogh NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  43. Matsushima KI, Sakagami JI (2013) Effects of seed hydropriming on germination and seedling vigor during emergence of rice under different soil moisture conditions. Am J Plant Sci 4:1584–1593CrossRefGoogle Scholar
  44. Metzner H, Ran H, Senger H (1965) Untresuchugen zur synchronisier barkit ein zeiner pigment Mangol Mutanten von chloreila. Planta 65:186CrossRefGoogle Scholar
  45. Mukherjee S, Mukherjee D (2015) Growth responses and changes in protein and nucleic acids of Cajanus cajan L. under normal, excess and deficient water supply. Int J Agric Innov Res 3:1574–1578Google Scholar
  46. Murkowski A (2001) Heat stress and spermidene affect chlorophyll fluorescence in tomato plants. Biol Plant 44:53–57CrossRefGoogle Scholar
  47. Nawaz A, Amjad M, Iqbal J (2009) Effect of thermal hardening on germination and seedling vigour of tomato. J Res (Science) Bahauddin Zakariya Univesity, Multan, Pakistan 10:39–49Google Scholar
  48. Pallet KE, Young AJ (1993) Carotenoids. In: Alscher RG, Hess JL (eds) Antioxidants in higher plants. CRC Press, Boca Raton, Ann Arbor, London, Tokyo, pp 60–89Google Scholar
  49. Rasheed R, Wahid A, Forooq M, Hussain I, Basra SMA (2011) Role of proline and glycinbetadine in improving heat tolerance of sprouting sugarcane (Saccharum sp.) buds. Plant Growth Regul 65:35–45CrossRefGoogle Scholar
  50. Reda F, Mandura HMH (2011) Response of enzymes activities, photosynthetic pigments, proline to low or high temperature stressed wheat plant (Triticum aestivum L.) in the presence or absence of exogenous proline or cysteine. Int J Acad Res 3:108–116Google Scholar
  51. Rehman AU, Farooq M, Ali H, Sarwar N, Qamar R (2014) Thermal hardening improves germination and early seedling growth of chickpea. Asian J Agri Biol 2:51–58Google Scholar
  52. Ruan YL, Jin Y, Yang YJ, Li GT, Boyer JS (2010) Sugar input, metabolism and signaling mediated by invertase: roles in development yield potential and response to drought and heat. Mol Plants 3:942–955CrossRefGoogle Scholar
  53. Sadak MS, Orabi SA (2015) Improving thermotolerance of wheat plant by foliar application of citric acid and oxalic acid. Int J Chem Tech Res 8:111–123Google Scholar
  54. Schmidt G, Thaunhauser SJ (1945) A method for determination of deoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J Biol Chem 161:83–89PubMedGoogle Scholar
  55. Selvaraj MG, Burow G, Burke JJ, Belankar V, Puppala N, Burow MD (2011) Heat stress secreening of peanut (Archis hypoguea L.) seedling for acquired thermotolerance. Plant Growth Regul 65:83–91CrossRefGoogle Scholar
  56. Shao HB, Chuc LY, Zang JH, Lua ZH, Hug YC (2007) Change of some anti-oxidative physiological induces under soil water difficit among 10 wheat (Triticum aestivum L.) genotypes at tillering stage. Colloids Surf B Biointerfaces 54:143–149CrossRefGoogle Scholar
  57. Solanki JK, Samangi SK (2014) Effect of drought stress on proline accumulation in peanut genotypes. Int J Adv Res 2:301–309Google Scholar
  58. Souza MR, Devaraj VR (2013) Induction of thermotolerance through heat acclimation in lablab bean (Dilichos lablab). Afr J Biotechnol 12:5695–5704Google Scholar
  59. Souza RP, Machadoa EC, Silva JAB, Lagoa AM, Silveira JAG (2004) Photosynthetic gas exchange chlorophyll fluorescence and some associated metabolic changes in cowpea (Vigna anguiculata) during water stress and recovery. Environ Exp Bot 51:45–56CrossRefGoogle Scholar
  60. Steel RG, Torrie JH (1966) Principles and procedures of statistics with special references to biological sciences. Mc.Craw- Hill Book Company- Inc- New York, Toronto- London, p 481Google Scholar
  61. Sujatha K, Sivasubramaniam J, Padma J, Selvarani K (2013) Seed hardening. Int J Agric Sci 9:392–412Google Scholar
  62. Taylor AG, Allen PS, Bonnett MA, Bradford KJ, Burris JS, Misra MK (1998) Seed enhancement. Seed Science Research 8(2):245–256.  https://doi.org/10.1017/S0960258500004141 CrossRefGoogle Scholar
  63. Troll W, Lidsley J (1955) Photometric methods for determination of proline. J Biol Chem 215:655–660PubMedGoogle Scholar
  64. Warrag MOA (1999) Flowering and fruiting of tomato (Lycopersicum esculentum Mill) in Qassin Saudi Arabia during summer. J King Saud Univ Agric Sci 3:241–250Google Scholar
  65. Winzler RJ (1955) Methods in biochemical analysis. Interscience, New York, p 270Google Scholar
  66. Yaish MW (2015) Proline accumulation is a general response to abiotic stress in the data palm tree (Phoenix dactylifera L.). Genet Mol Res 14:9943–9950CrossRefGoogle Scholar
  67. Zhou R, Kjaer KH, Rosenqvist E, Yu X, Wu Z, Ottosen CO (2017) Physiological response to heat stress during seedling and anthesis stage in tomato genotypes differing in heat tolerance. J Agron Crop Sci 203:68–80CrossRefGoogle Scholar

Copyright information

© The Author(s) 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of BotanyNational Research CentreCairoEgypt
  2. 2.Department of Botany, Girls College of ScienceDamam UniversityDamamSaudi Arabia

Personalised recommendations