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. 2021 Mar 5;27(3):469–481. doi: 10.1007/s12298-021-00958-8

Impact of melatonin and tryptophan on water stress tolerance in white lupine (Lupinus termis L.)

Mervat Shamoon Sadak 1,✉, Amany Abd El-Mohsen Ramadan 1
PMCID: PMC7981349  PMID: 33854277

Abstract

Melatonin  has been identified as a signal molecule that regulates plant responses to different abiotic and biotic stresses. Melatonin (MT) and its precursor tryptophan (Try) have a major role in improving plant stress tolerance to different environmental stresses such as water deficiency. The rapid increase in the Egyptian population caused insufficient protein sources, especially those of animal origin, in their diet. The possible solution is to augment the diet with legumes such as white lupine which are relatively rich in protein. Thus, the current experimental work was carried out to find changes in growth, biochemical aspects and yield quantity and quality of white lupine plant with spraying of both MT and Try at different concentrations on plant shoot under water deficit stress conditions. Results showed that water deficit (75 or 50% of water irrigation requirements; WIR) caused significant reduction in growth, photosynthetic pigments, indole acetic acid and yield compared with those received 100% WIR. Seed yield significantly decreased (p < 0.05) by 26.98 and 41.64% by decreasing WIR to 75 and 50%. The decrease was accompanied by significant increase in phenolic contents, hydrogen peroxide, lipid peroxidation and some antioxidant enzymes, while nitrate reductase enzyme was decreased. However, external application of either MT or Try significantly alleviated the adverse effects of water deficit (growth suppression), since MT or Try-treated plants recovered more quickly than untreated plants. Moreover, MT or Try—treated plants had higher photosynthetic pigments, indole acetic acid, phenolic, as well as yield quantity and quality under the three WIR as compared with untreated plants. Melatonin treatment at 100 µM and Tryptophan at 200 µM increased weight of seeds/plant by 78.29 and 52.19%, 71.49 and 43.78% and 41.21 and 13.07% in plants irrigated with 100, 75 and 50% WIR, respectively. Exogenous MT and Try significantly reduced hydrogen peroxide and malondialdehyde content, while markedly increased the activities of antioxidant enzymes and nitrate reductase under different WIR. Finally, the current study concluded that MT and Try treatments alleviated the detrimental effects of water deficiency and accelerated the recovery mainly via improving white lupine plants tolerance in forms of enhancing photosynthetic pigments, indole acetic acid, phenolic and antioxidant capacity.

Keywords: Melatonin, Tryptophan, Yield, Amino acids, Antioxidant enzymes, Lupinus termis

Introduction

White lupine (Lupinus termis L.) is one of the eldest field crops cultivated in a wide range of habitats as Mediterranean, North and Eastern Africa (Lucas et al. 2015). The nutritional value of white lupines seeds is similar to soybean seeds and superior to seeds from other leguminous plants with respect to protein content. White lupine seeds are valuable source of protein and oil (Aiking 2011). The seeds contain 5–13% oil, 33–40% crude protein high in essential amino acid like cystine, methionine, phenylalanine, tyrosine, threonine, valine leucine isoleucine and lysine (Písaříková and Zralý 2009). Due to the presence of high antioxidants content (α- and δ-) tocopherol (5.41 and 4.23 mg/100 g oil, respectively) white lupine seed oil quality is used in different food products (El-Awadi et al. 2016).

Like other legumes, white lupine plants can fix atmospheric nitrogen in the soil, thus increasing its fertility, permeability and water storage (Wolko et al. 2011). Therefore, the plant could be cultivated for green manure also. White lupine plant’s development proceeds either through vertical expansion (using growth promotors as antioxidant vitamins, amino acids etc.) or horizontal expansion (cultivation in sandy soils). Water scarcity is one of those problems which has adversely affected plant growth and productivity (Zhang and Huang 2013). Tolerance and growth of plants under water deficit conditions depend on the interactions between morpho-physiological, anatomical and biochemical variations (Moschen et al. 2017). Water deficit effect on different plants varies depending on the growth stage of plant, and duration of stress (Khan et al. 2017). For enhancing plant tolerance to water deficit, different natural compounds are used through external applications either as seed priming or foliar applications on various plant species. Advanced agronomic cultural practices were made using efficient, economic and inexpensive compounds. These compounds might be plant hormones, biostimulators (e.g. amino acids) or other signal molecules (Sadak 2016; Sadak et al. 2020a), from which melatonin and its precursor tryptophan has been chosen.

Melatonin (N-acetyl-5-methoxy tryptamine) MT is a new efficient plant growth regulator used on different crops to tolerate environmental stress. It is widely present within wide range of concentrations in plant and other living organisms (Tan et al. 2012; Posmyk and Janas 2009). The lipophilic and hydrophilic nature of melatonin facilitates its rapid transport through morpho-physiological obstructions to reach inside the plant cells (Tan et al. 2012). Melatonin is involved in the regulation of physiological processes such as ion homeostasis (Park et al. 2013). Moreover, melatonin has an important dual function as a stress defender (Manchester et al. 2000) and as an antioxidant and an effective free radical scavenger in plants (Zhang et al. 2015; Wang et al. 2012; Tan et al. 2002).

L-tryptophan (Try) is one of most known amino acids for plant growth under normal environmental conditions as well as under different abiotic stresses. In higher plants, L-Tryptophan is a physiological precursor of melatonin (Chen et al. 2009). It plays an important role as an osmolyte compound, can regulate transport of various ions as nutrients, stomatal opening modulator and help in reducing the toxic effects of heavy metals (Rai 2002). Moreover, tryptophan could enhance various biochemical processes via regulating plant growth, differentiation and improving the ability to obtain nutrients and water (Talaat et al. 2005; Dawood and Sadak 2007).

Considering all these points, the aim of this investigation, was to study the physiological role of melatonin and tryptophan in alleviating the reducting effect of different water deficit conditions on growth, yield and quality attributing traits of white lupine plant.

Materials and methods

Experimental procedure

Two pot experiments were conducted in the greenhouse of National Research Centre, Dokki, Cairo, Egypt (30° 3′0″ N/31°15′0″ E), during two winter season of 2017/2018 and 2018/2019 (from 7th November to 22th April). Daytime temperature ranged from 14.5 to 30.2 °C with an average of 23.2 ± 3.8 °C, whereas temperature at night were 12.4 ± 1.8 °C, with minimum and maximum of 8.0 and 17.6 °C, respectively. Daily relative humidity averaged 57.7 ± 9.6% in a range from 38.1 to 78.7%. White lupine (Lupinus termis L.) variety Giza 2 were obtained from Agricultural Research Centre, Ministry of Agriculture and Land Reclamation, Egypt. To study the effect of foliar treatment of melatonin (0, 50, 100 or 150 µM) and tryptophan (0, 100, 200 or 300 µM) (chosen according to preliminary experiment) on white lupine plant grown under different water irrigation conditions (100, 75 or 50% WIR). White lupine seeds were selected for uniformity (by choosing those of equal size and with the same color). The selected seeds were washed with distilled water, sterilized with 1% sodium hypochlorite solution for about 2 min and thoroughly washed again with distilled water. Five uniform air dried white lupine seeds were sown along a central row in each pot at 30-mm depth in plastic pots, each filled with about 7 kg clay soil from Giza, consisting of the upper 10 cm of soil collected from an area of undisturbed native vegetation. To reduce compaction and improve drainage, the soil was mixed with yellow sand in a proportion of 3:1 (Wt:Wt). Granular ammonium sulphate with 20.5% nitrogen (N) at a rate of 40 kg N ha−1 and single superphosphate (15% P2O5) at a rate of 60 kg P2O5 ha−1 were added to each pot. The N and phosphorus (P) fertilizers were mixed thoroughly into soil of each pot immediately before sowing. Soil field capacity in the pots was estimated by saturating the soil in the pots with water and weighing them after they had drained for 48 h. Foliar spraying of white lupine plants with different concentrations of either ME or Try was done at 30 and 45 days from sowing. The three water irrigation requirement levels used were 100, 75 and 50%. The pots were irrigated with equal volumes of the WIR levels at 50 days after sowing.

Plant samples were taken 75 days after sowing for measurements of growth characters as shoot length (cm), leaves (number/plant), fresh and dry shoot weight (g/plant). At harvest, yield and yield components (number of pods/plant, weight of pods and seeds/plant, number of seeds/plant and weight of 100 seeds) were recorded.

Chemical analysis

Plant samples were taken for chemical analysis of photosynthetic pigments, chlorophyll a and b and carotenoids contents in fresh leaves were estimated using the method of Lichtenthaler and Buschmann (2001). Indole acetic acid content was extracted and analyzed by the method of Larsen et al. (1962). Phenolic content was measured as described by Danil and George (1972). Total carbohydrates determined according to Herbert et al. (1971). The levels of lipid peroxidation was measured by determining the levels of malondialdehyde (MDA) as a product of lipid peroxidation assayed by thiobarbituric acid reactive substrates (TBARS) contents using the method of Hodges et al. (1999). Hydrogen peroxide (H2O2) concentration was determined according to Velikova et al. (2000).

Enzyme extractions as well as assay of superoxide dismutase were carried out according to the method of Chen and Wang (2006). Peroxidase (POX, EC 1.11.1.7) activity was assayed by the method of Bergmeyer (1974). Catalase (CAT, EC 1.11.1.6) activity was determined spectrophotometrically by following the decrease in absorbance at 240 nm (Chen and Wang 2006). The enzyme activities were calculated by Kong et al. (1999). The activity of nitrate reductase (NR, EC 1. 7. 1. 1) was measured according to Jaworski (1971) and expressed as nano moles of nitrite produced per gram fresh weight per hour (nM NO2/g FW/h). Seeds after harvest were used to determine chemical composition viz carbohydrates, oil and protein contents as well as amino acid constituents. The protein content was determined by Micro-kjeldahl method (AOAC 1990). The oil content of lupine seeds was extracted according to Kates and Eberhardt (1957). Identification and determination of the amino acid composition of the yielded seeds was carried out by using HPLC (Eppendorf, Germany) according to Gehrke et al. (1985).

Statistical analysis

The experiment was conducted in a completely randomized design (CRD) with 5 replicates. Data on morphological, physiological and yield characters and seed quality were subjected to conventional methods of analysis of variance according to MSTAT-C (1988) statistical analysis program. Means were compared by using Least Significant Difference (LSD) test at 5% probability level.

Results

Growth parameters

Regarding vegetative growth parameters (shoot length, number of leaves/plant, fresh and dry weights of shoot) of white lupine plants were significantly (p < 0.05) decreased by 18.43 & 26.26 in shoot length, 18.89 and 40.55 in number of leaves/plant, 17.72 & 41.53 in fresh weights and 18.60 & 42.44 in dry weights of shoot with decreasing WIR from 75 to 50%, respectively compared to those irrigated with 100% WIR (Table 1). Whereas, all external treatments of either MT (50, 100 or 150 µM) or Try (100, 200 or 300 µM) significantly (p < 0.05) increased the measured growth parameters of white lupine plants via alleviating the reducing effects of water deficit thus significantly increased the above mentioned parameters in most treatments as compared to their corresponding controls (Table 1). Melatonin external treatments were more pronounced than tryptophan treatments in terms of vegetative growth parameters.

Table 1.

Effect of different concentrations of melatonin or tryptophan on growth traits of white lupine plants under different water irrigation requirements

WIR (%) Treatment materials Conc. (µM) Shoot length (cm) Number of leaves/plant Shoot fresh wt. (g) Shoot dry wt. (g)
100 Control 0 34.33de 12.33def 8.86 cd 1.72d
Melatonin 50 39.33b 17.33a 10.35b 1.89ab
100 42.00a 18.33a 11.76a 2.05a
150 40.33b 14.67bc 9.98b 1.95bc
Tryptophan 100 36.00c 14.67bc 10.14b 1.72c
200 39.33b 15.67b 10.18b 1.91ab
300 39.33b 15.00bc 9.88b 1.79c
75 Control 0 28.00g 10.00gh 7.29f 1.40
Melatonin 50 33.00e 11.00efgh 8.42de 1.57
100 36.33c 13.33cd 9.08c 1.81
150 33.33de 12.67de 7.90e 1.63
Tryptophan 100 31.00f 11.00efgh 7.96e 1.60
200 31.33f 12.67de 8.70 cd 1.67
300 34.67d 11.33efg 8.54 cd 1.60
50 Control 0 25.33i 7.33i 5.18 h 0.99
Melatonin 50 27.67gh 9.33h 6.19 g 1.19
100 30.67f 10.00gh 6.28 g 1.39
150 26.67ghi 11.67defg 6.31 g 1.31
Tryptophan 100 27.00gh 10.67fgh 5.94 g 1.03
200 26.67ghi 12.33def 6.15 g 1.23
300 26.33hi 10.67fgh 6.01 g 1.30
LSD (5%) 1.31 1.55 0.51 NS
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Mean values with the same superscripts are not significantly different at p ≤ 0.05

WIR, water irrigation requirements; NS, not significant

Photosynthetic pigments

Water deficit had a significant influence on white lupine leave’s content from the studied photosynthetic pigments i.e. chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids as well as total pigments (Table 2). Decreasing water irrigation requirement (increasing water deficit) from 100 to 75 or 50% has reduced gradually Chl a, Chl b, carotenoids and total pigments. Foliar treatment of white lupine plants with either MT at 50, 100 or 150 µM or Try at 100, 200 or 300 µM have significantly (p < 0.05) increased Chl a, Chl b, carotenoids and total pigments. Melatonin treatment with different concentrations were observed as more effective than tryptophan and induced a significant increase in chlorophyll a, b, carotenoids and total pigments (Table 2). Moreover, 100 µM melatonin was the most pronounced treatment at different WIR levels.

Table 2.

Effect of different concentrations of melatonin or tryptophan on photosynthetic pigments (mg/g fresh weight) of white lupine plants under different water irrigation requirements

WIR (%) Treatment materials Conc. (µM) Chl a Chl b Carotenoids Total pigments
100 Control 0 1.26d 0.913g 0.592g 2.77d
Melatonin 50 1.38ab 0.993c 0.647c 3.02ab
100 1.42a 1.033a 0.669a 3.13a
150 1.34bc 0.970d 0.629d 2.94bc
Tryptophan 100 1.32c 0.954f 0.622e 2.90c
200 1.39ab 1.012b 0.653b 3.06a
300 1.32c 0.955e 0.620f 2.89c
75 Control 0 0.98hi 0.713n 0.459n 2.15hij
Melatonin 50 1.07fg 0.784k 0.504k 2.36fg
100 1.18e 0.854h 0.554h 2.59e
150 1.11f 0.803j 0.521j 2.43f
Tryptophan 100 1.03gh 0.745m 0.483n 2.26gh
200 1.06fg 0.768l 0.498l 2.33fg
300 1.17e 0.845i 0.548i 2.56e
50 Control 0 0.87j 0.632t 0.410s 1.91k
Melatonin 50 0.96i 0.697p 0.452o 2.11ij
100 0.98hi 0.712n 0.462n 2.16hi
150 0.97hi 0.704o 0.457n 2.13ij
Tryptophan 100 0.93i 0.671s 0.435r 2.03j
200 0.94i 0.680r 0.441q 2.06ij
300 0.95i 0.686q 0.445p 2.08ij
LSD (5%) 0.05 0.002 0.002 0.10
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Mean values with the same superscripts are not significantly different at p ≤ 0.05

WIR, water irrigation requirements

Biochemical components

The two used water irrigation requirements (75 and 50% WIR) have gradually and significantly decreased indole acetic acid contents compared to control (Table 3) of white lupine. Meanwhile, phenolic compounds, hydrogen peroxide and lipid peroxidation (as MDA) have gradually and significantly increased by decreasing water irrigation requirement compared to unstressed white lupine plants. However, all treatments of MT or Try caused significant increase in IAA and phenolic contents accompanied by significant decrease in H2O2 and MDA contents in plants irrigated with 100% WIR compared with control plants (Table 3). Moreover, under water deficit conditions, foliar treatment of either MT or Try gave more or less similar effect on some biochemical components in treated plants and those irrigated with 100%WIR as well. It is clear from Table 3 that, 100 µM MT was the most effective treatment either for enhancing some parameters (IAA and phenolic) or inhibition of other tested ones (H2O2 and MDA).

Table 3.

Effect of different concentrations of melatonin or tryptophan on biochemical constituents of white lupine plants under different water irrigation requirements

WIR (%) Treatment materials Conc. (µM) IAA (µg/g) Phenols (mg/100 g) H2O2 MDA
(n mole/g fresh wt.)
100 Control 0 33.25i 54.00o 6.57hi 16.11j
Melatonin 50 40.93f 63.19l 6.08jk 14.48k
100 51.78a 68.26ij 5.83k 11.94m
150 45.60d 66.91k 6.07jk 13.54l
Tryptophan 100 41.93e 60.69n 6.00ik 13.94kl
200 49.17b 63.24l 6.30ij 12.47m
300 46.46c 62.10m 6.09jk 12.07m
75 Control 0 23.41no 63.94l 9.16e 20.74g
Melatonin 50 30.92k 69.15h 7.08g 18.64h
100 35.84g 72.70e 6.65hi 17.56i
150 31.95j 68.94hi 6.89gh 18.70h
Tryptophan 100 31.04k 67.64j 8.10f 19.16h
200 34.70h 71.03f 7.74f 17.76i
300 33.07i 69.91g 7.19g 18.70h
50 Control 0 18.89p 70.73f 12.67a 30.04a
Melatonin 50 22.76o 76.74c 10.77b 26.55d
100 29.10l 86.65a 9.76d 22.03f
150 28.90q 80.50b 9.72d 23.57e
Tryptophan 100 23.84n 72.81e 10.41bc 27.39c
200 26.84m 73.70d 10.28c 28.40b
300 26.94m 76.74c 10.41bc 26.56d
LSD (5%) 0.81 0.72 0.38 0.55
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Mean values with the same superscripts are not significantly different at p ≤ 0.05

WIR, water irrigation requirements

Changes in antioxidant enzymes

The antioxidant enzyme activities of SOD, CAT, POX and NR in shoots of white lupine plants were significantly increased in response to moderate and high water deficit (75 and 50% WIR) compared with the control plants (Table 4). In addition, different concentrations of melatonin or tryptophan significantly increased the studied enzymes (SOD, CAT and POX) accompanied with significant decrease in NR enzyme (Table 4) compared to their corresponding control under unstressed and water deficit conditions.

Table 4.

Effect of different concentrations of melatonin or tryptophan on some antioxidant enzymes (Unit/g fresh wt./min) of white lupine under different water irrigation requirements

WIR (%) Treatment materials Conc. (µM) SOD CAT POX NR (nM NO/g fresh wt./h)
100 Control 0 18.15s 23.99p 347.50q 324.60gh
Melatonin 50 26.70q 28.31n 376.45o 313.53j
100 32.01n 36.07l 402.85m 270.50p
150 36.66l 30.87m 406.40l 286.00m
Tryptophan 100 25.57r 26.95o 372.99p 282.35n
200 27.38pq 35.62l 383.54n 266.50q
300 31.000 30.74m 374.84op 274.52o
75 Control 0 28.01p 40.92k 474.50k 341.86c
Melatonin 50 35.16m 49.90j 509.50i 322.44hi
100 42.25k 59.06e 523.94g 302.33l
150 50.91l 52.89h 513.31h 331.90e
Tryptophan 100 36.89l 49.21j 503.50j 312.65j
200 51.83g 56.99f 513.64h 304.99k
300 48.26i 54.01g 524.15g 325.61fg
50 Control 0 44.94g 51.84i 563.90f 351.45a
Melatonin 50 56.56c 61.67d 568.17e 326.15fg
100 60.23a 68.81a 573.34d 313.01j
150 54.09e 63.43c 613.35e 336.08d
Tryptophan 100 52.96f 59.31e 674.45a 321.01i
200 58.14b 64.23b 652.45b 327.69f
300 55.02d 62.32c 613.45c 345.23b
LSD (5%) 0.79 0.75 2.91 2.38
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Mean values with the same superscripts are not significantly different at p ≤ 0.05

WIR, water irrigation requirements

Yield and its components

The traits recorded at harvest (Table 5) which included yield and its components and chemical constituents such as percentages of protein, carbohydrates and oil of white lupine plant showed gradual decrease in water deficit of 75 and 50% WIR in compare to 100% WIR. On the other hand, external treatments of white lupine with all concentrations of melatonin (50, 100 or 150 µM) or tryptophan (100, 200 or 300 µM) increased yield and its components as well as total carbohydrate (TCH), oil and protein percentages of the yielded seeds in plants irrigated with 100% WIR and those subjected to water deficit requirements (75 and 50% WIR) as well significantly (p < 0.05). Data clearly showed that 100 µM melatonin was the most effective treatment in increasing weight of seeds/plant followed by 200 µM tryptophan treatment in most studied yield parameters.

Table 5.

Effect of different concentrations of melatonin or tryptophan on yield and yield components of white lupine plants under different water irrigation requirements

WIR (%) Treatment materials Conc. (µM) No. of pods/plant Wt. of pods/plant Weight of seeds/plant No. of seeds /plant Weight of 100 seeds TCH% Oil% Protein%
100 Control 0 3.67 4.40fgh 3.41h 11.00fgh 28.15 41.29 9.85h 29.02h
Melatonin 50 4.67 6.42c 5.17b 15.33a 32.31 43.08 10.13h 31.07f
100 5.33 8.67a 6.08a 13.33bc 36.90 44.61 11.42g 33.34b
150 5.00 7.38b 5.15b 12.33cde 33.07 43.75 11.19 g 32.69 cd
Tryptophan 100 4.67 5.34de 4.60d 13.00bc 31.03 42.59 9.99 h 30.74 g
200 4.67 6.74bc 5.19b 15.67a 35.70 43.75 10.01 h 31.74e
300 5.33 5.98cd 4.92c 13.67b 33.39 42.86 9.84 h 31.10f
75 Control 0 2.33 3.35ij 2.49j 8.33k 21.05 39.55 11.24 g 30.69 g
Melatonin 50 3.33 4.88ef 3.71fg 9.67ij 24.39 40.47 12.06ef 32.74 cd
100 3.67 6.06cd 4.27e 11.67def 28.71 41.34 12.85b 33.59ab
150 3.67 5.44de 3.83f 11.33efg 25.55 40.97 12.20de 32.70 cd
Tryptophan 100 3.67 4.79ef 3.28h 12.67bcd 28.14 40.86 11.78f 31.10f
200 3.67 4.95ef 3.58g 13.33bc 25.40 41.06 12.06ef 32.74 cd
300 3.67 4.64efg 3.29h 11.67def 23.08 40.93 11.96ef 33.74a
50 Control 0 2.67 3.12j 1.99l 7.00l 15.76 38.40 12.89b 31.75e
Melatonin 50 3.33 3.49ij 2.28k 8.67jk 18.29 39.55 13.27a 32.76 cd
100 3.67 4.17fghi 2.81i 10.67fghi 20.97 40.24 12.77b 33.87a
150 4.00 4.16fghi 2.59j 10.00hi 20.21 39.84 12.44 cd 32.90c
Tryptophan 100 3.67 3.77ghij 2.32k 10.33ghi 17.65 39.27 12.59bc 32.66 cd
200 3.67 3.50ij 2.25k 11.67def 18.69 40.04 12.65bc 32.40d
300 4.00 3.70hij 2.22k 10.33ghi 17.82 39.94 12.39 cd 32.90c
LSD (5%) NS 0.78 0.14 1.03 NS NS 0.29 0.033
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Mean values with the same superscripts are not significantly different at p ≤ 0.05

WIR, water irrigation requirements; NS, not significant

Amino acid constituents of white lupine seeds

The amino acids constituents of the yielded seeds of white lupine plants treated with melatonin or tryptophan grown under different water irrigation requirements i.e. normal irrigation (100% WIR) and water deficit (75 and 50% WIR) are presented in Table 6. Data showed that amino acids of white lupine seeds consists of 17 amino acids with low contents of sulphur-containing amino acids (cystine & methionine). Total aromatic amino acids (phenylalanine, tyrosine, histidine and tryptophan) present with higher amount than total sulphur containing amino acids. With respect to the non-essential amino acids, glutamic acid was the most abundant one followed by arginine and aspartic acid. Proline content in white lupine seeds increased in the treated plants. Herein, moderate (75% WIR) and higher (50% WIR) water deficit decreased ESS/TAA ratio compared with control. Meanwhile, treatments of both melatonin and tryptophan increased ESS/TAA percentage under different irrigation requirements. Water deficit markedly increased total amino acid contents, total essential amino acid and total non-essential amino acid contents. Meanwhile, total essential AA/total non-essential AA ratio decreased as compared to the control plants. The melatonin or tryptophan treatments, in most cases, increased the content of total amino acids, essential amino acids and the ratio of essential to non-essential amino acids were noticed compared to those under water deficit conditions (Table 6).

Table 6.

Effect of melatonin (100 µM) or tryptophan (200 µM) on amino acid constituents of the yielded seeds of white lupine plants under different water irrigation requirements

WIR (%)
Treatments
Amino acid		 100 75 50
Control MT Try Control MT Try Control MT Try
Cystine* 0.753 0.725 0.767 0.767 0.757 0.705 0.712 0.732 0.742
Methionine* 0.409 0.505 0.548 0.381 0.363 0.435 0.264 0.314 0.236
Total sulfur AA 1.162 1.230 1.315 1.148 1.120 1.140 0.976 1.046 0.978
Phenylalanine* 1.839 2.368 2.427 2.265 2.395 2.335 2.223 2.275 2.395
Tyrosine* 1.729 1.975 1.795 1.975 1.945 2.168 1.324 1.523 1.665
Histidine 0.992 0.768 0.965 1.165 1.368 1.565 1.532 1.453 1.752
Tryptophan 0.132 0.451 0.565 0.363 0.732 0.634 0.421 0.527 0.569
Total aromatic AA 4.692 5.562 5.752 5.768 6.440 6.702 5.500 5.778 6.381
Threonine* 1.727 1.849 1.968 1.965 1.968 2.152 2.275 2.243 2.262
Valine* 1.379 1.762 1.884 1.264 1.468 1.485 1.635 1.725 1.678
Leucine* 2.346 2.675 2.765 2.568 2.268 2.395 2.314 2.758 3.154
Isoleucine* 1.574 2.495 2.264 1.868 2.168 1.252 1.615 2.324 2.845
Lysine* 2.034 2.325 3.195 2.775 3.152 3.352 3.152 3.154 3.624
Essential Amino Acids (ESS AA) 13.79 16.68 17.61 15.83 16.48 16.28 15.51 17.05 18.60
Aspartic acid 3.318 3.685 3.565 3.665 2.985 2.935 3.231 2.652 2.352
Serine 1.963 2.013 2.168 2.168 2.284 2.269 3.654 3.475 3.985
Glutamic 7.875 7.149 7.465 8.568 7.768 8.168 8.652 7.514 7.652
Proline 2.326 3.563 3.965 3.668 3.565 3.535 5.352 5.351 5.625
Glycine 1.552 2.259 2.265 2.362 2.768 2.668 2.356 2.758 2.625
Alanine 1.152 1.585 1.984 2.265 2.568 2.968 3.352 3.744 3.625
Arginine 4.235 4.952 4.658 6.168 5.268 5.385 7.625 9.854 9.485
Non-essential AA (non-ess AA) 28.24 31.98 33.35 36.16 35.75 36.83 41.67 43.11 44.05
ESS AA/non-ess AA 0.488 0.521 0.528 0.438 0.461 0.442 0.372 0.395 0.422
Total amino acids (TAA) 42.03 48.66 50.96 51.99 52.23 53.11 57.18 60.16 62.65
ESS/TAA 32.81 34.27 34.56 30.45 31.56 30.65 27.13 28.34 29.69
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*Denotes to essential amino acids

Discussion

Changes in growth parameters

Data of various growth traits showed their reduction under water deficit conditions (Table 1). The most important reason for this decrease might be related to low water uptake, disturbance in photosynthesis, reduced endogenous hormone biosynthesis and decreased activity of different important enzymes (Naghizadeh et al. 2019). Gholami et al. (2010) stated that water deficiency among the environmental stresses (oxidative stress) adversely affects plant growth, organs and activity via accumulation of reactive oxygen species (ROS) and it was removed by antioxidant defense system through decreasing the toxic effects of stress. Foliar treatment of either melatonin or tryptophan with different concentrations caused significant (p < 0.05) increase in the studied growth parameters under different irrigation requirements (normal and water deficit conditions) (Table 1). Li et al. (2015) stated that external treatment of melatonin and its precursor tryptophan which are known as a natural, cheap and safe substance for the environment could improve plant tolerance to water deficit conditions. Similar results were reported in studies by Kabiri et al. (2018) on Moldavian balm, Huang et al. (2019) on maize and Sadak et al. (2020b) on Moringa oleifera using melatonin. While, treatment of chickpea with L-tryptophan was fruitful for improving their growth (Abbas et al. 2013; El-Awadi et al. 2017). The noticed effect of tryptophan resulted via the biosynthesis of indole acetic acid as auxin hormone and consequently improved plant growth (Mohite 2013).

Changes in photosynthetic pigments

Decreasing water irrigation requirement or water deficit significantly (p < 0.05) decreased photosynthetic pigment content of white lupine plants (Table 2). Meanwhile, melatonin and tryptophan at different concentrations increased photosynthetic constituents under different irrigation requirement. This is in agreement with Naghizadeh et al. (2019), Sadak and Bakry (2020) and Sadak et al. (2020b), who showed that drought stress reduced photosynthetic pigments of Dracocephalum moldavica, flax and wheat plants, respectively. The reduction in photosynthetic pigments under water deficit conditions might be related to the destruction of chloroplast and photosynthetic apparatus, chlorophyll, photo-oxidation induced breakdown of chlorophyll biosynthesis precursors, hormonal imbalance and activation of chlorophyll degrading enzymes (Wang et al. 2013). Moreover, Sultana et al. (1999) reported that in water deficit plants, the decrease in carotenoids might be related to the destruction in β-carotene or formation of zeaxanthin in the xanthophyll cycle. Melatonin caused delaying of chlorophyll degradation of barley and leaf senescence compared to control via the enhancement of reactive oxygen species–scavenging enzyme activities and improved antioxidant contents (Arnao and Hernandez-Ruiz 2009). Moreover, the photosynthetic efficiency of many plants increased, since melatonin molecule suppressed the up-regulation of senescence-associated gene and significantly reduced chlorophyll degradation (Tan et al. 2012). Concerning the promoting role of tryptophan treatment, Barazan and Friedman (2004) reported that tryptophan affected the biosynthesis of chloroplast.

Indole acetic acid

Indole-3-acetic acid (IAA), the most common naturally occurring auxin, is a hormone produced by plants, fungi and bacteria. IAA plays a central role in modulating plant growth and development (Woodward and Bartel 2005; Teale et al. 2006). Tryptophan (Try) is a main precursor for IAA biosynthesis in microbes. The reduction in indole acetic acid (IAA) contents in white lupine plants irrigated with moderate (75%) and low (50%) water irrigation requirements (water deficit) and were accompanied with reduced vegetative growth parameters (Table 3). The decreased levels of IAA under water deficit stress could be referred to the inhibition in IAA biosynthesis and/or increased their breakdown or transfered to inactive form. Kazan (2013) considered the reduction in auxin contents under water deficiency stress-responsive transcription factor responsible for modification in auxin and root development environment. On the contrary, the positive effect of both melatonin and tryptophan on IAA contents was similar to the results reported by Dawood and El-Awadi (2015) on faba plant and Sadak et al. (2020b) on Moringa oleifera plant.

Phenolic

The phenolic contents of white lupine leaves (Table 3) were significantly increased due to water deficit and/or exogenous application of melatonin or tryptophan. This increase in phenol contents can mitigate harmful effects of water deficit. Water deficit mediated disruptions in the biochemical processes causing an increase in phenolic compounds biosynthesis (Keutgen and Pawelzik 2009). In fact, the high level of reactive oxygen species in plants subjected to water deficit is commonly coincided by modifications in the net carbon acquisition that may have a profound effect on the signaling pathways of secondary organic compounds, especially leaf polyphenols (Radi et al. 2013). Phenolic substances have a significant effect as an antioxidant in scavenging reactive oxygen species (Huang et al. 2005). The positive role of either melatonin or tryptophan could be attributed to its signaling mechanism by triggering different metabolic pathways and stimulating the development of various substances (Tan et al. 2012). Sadak et al. (2020a, b) indicated that melatonin treatment of Moringa oleafera plants improved plant tolerance and increased the production of phenolic compounds under drought stress.

Hydrogen peroxide (H2O2) and lipid peroxidation

The data obtained in Table 3 induced a significant increase in H2O2 and lipid peroxidation (MDA content) due to the gradual reduction in irrigation water. The harmful effects of water deficit mitigated by foliar spraying of both melatonin or tryptophan. In this regard, water deficit among various environmental stresses stated earlier for increasing overproduction of free radical molecules (ROS) (Ozgur et al. 2013). These ROS molecules cause cell injury and increased leakage of electrolytes (Shi et al. 2007). Lipid peroxidation, a non-enzymic auto-oxidation process caused by over accumulated ROS, is generally seen as a predictor of oxidative stress caused by water deficit stress and plant susceptibility (Ozgur et al. 2013). Dawood and Sadak (2014) stated that subjecting canola plants to drought stress increased hydrogen peroxide and MDA contents. The increased levels of both H2O2 and MDA might be due to inadequate induction of antioxidant system. While, treating white lupine plants with melatonin and tryptophan lowered H2O2 and MDA contents. Earlier studies have reported that melatonin and its precursor tryptophan function as a lipid peroxidation inhibitor may be linked to its ability to react with radicals of lipid alcoxyl and peroxyl and disrupt the peroxidation chain (Zhang et al. 2013). Also they added that, a defensive effect of melatonin against H2O2 and MDA content reduction was documented under water deficit.

Changes in antioxidant enzymes

Antioxidant enzymes SOD, CAT, POX and NR were significantly (p < 0.05) increased due to moderate and severe water deficit relative to control plants (Table 4). In this concern, Gharache et al. (2013) showed the enhancing activities of SOD and APX which stimulate scavenging of reactive oxygen species (ROS), so conferring more drought resistance to plants. Sadak et al. (2019) stated that the increase in SOD and POX activities linked with protection from oxidative damage resulted by drought stress in flax cultivars. Superoxide is converted by the SOD enzyme to H2O2 (Noctor and Foyer 1998), thus scavenged to O2 and water by the antioxidant enzymes such as CAT and POX (Ozkur et al. 2009). The increased activity of CAT revealed drought tolerance in some canola cultivars (Omidi 2010). Some enzyme activities particularly nitrate reductase (NR) declined dramatically by increasing water deficit (Dawood and Sadak 2014). Moreover melatonin or tryptophan treatments improved different antioxidant enzymes (Table 4). Melatonin as well as its precursors tryptophan are known as endogenous scavenger of reactive oxygen species and also antioxidants (Zhang et al. 2013). These substances can directly capture ROS such as H2O2 (Cui et al. 2017). Li et al. (2017) concluded that melatonin, a signal of powerful lengthy-distance, can be transferred from treated plant organs to far untreated ones through vascular bundles, resulting in abiotic stress tolerance. Moreover, melatonin addition activates the antioxidant enzymes by increasing the transcription and activity levels (Zhang et al. 2014). In plant stress tolerance, the key function of melatonin considered enhancing plant antioxidant defense systems (Zhang et al. 2015). Nitrate reductase activity, as a key to biosynthetic enzymes for NO generation, was significantly decreased in treated plants compared with stressed ones which supported the decline in NO content (Antoniou et al. 2017).

Changes in yield and its components

Table 5 shows that, water stress decreased yield, its components and chemical constituents of the yielded seeds of white lupine. It is worth mentioning that the existence of water to plant in different stages of growing influences their development and growth. The decrease in the yield of white lupine plants resulted from the reduction in growth traits (Table 1) and pigment's content (Table 2). The reducting effect of drought stress on yield are in accordance with Sadak and Bakry (2020) on flax plants. The reductions in photosynthetic pigment contents in water deficit stressed plants of white lupine leaves induced photosynthetic enzyme activities inhibition, consequently decreased carbohydrate accumulation in mature leaves thus reduced the rate of carbohydrate transfer to developing organs (Anjum et al. 2003). Meanwhile, all applied treatments of melatonin or tryptophan caused significant increase in yield components. Melatonin mediate growth process and ion balance and increased the vegetative growth and yield parameters of different plant species (Sarropoulou et al. 2012). The increase in the yield of plants as a result of tryptophan application attributed to its positive effect on the cellular division and the production of phyto-hormones, e.g., auxin (Abbas et al. 2013). Moreover, El-Bassiouny (2005) added that tryptophan enhanced nutrients uptake, chlorophyll and protein synthesis. Dawood and Sadak (2007) attributed the increase in seed weight with tryptophan treatments to the increased amount and translocation of assimilates to the produced seeds. The tested treatments (as shown in Table 5) caused significant increase in the nutritional value of white lupine plant’s yielded seeds (carbohydrate, oil and protein percentages). El-Awadi et al. (2017) found that total carbohydrate and oil were increased in chickpea plants in response to tryptophan treatment. Moreover, the obtained effect of tested amino acids on total carbohydrate contents may be attributed to their role in the biosynthesis of chlorophyll. The positive effect of melatonin and tryptophan on photosynthetic pigments (Table 2) supposed to be associated with the overproduction of total carbohydrates and the increase of yield quality and quantity.

Changes in amino acid constituents

Amino acids content (Table 6) showed that melatonin or tryptophan, in most cases, increases the content of total amino acids, essential amino acids and the ratio of essential to non-essential amino acids in plants under water deficit conditions. Amino acid constituents of plants are changeable in response to environmental stress (Abd Elhamid et al. 2014). The increased essential amino acids proportion raises the biochemical quality of seeds. The provisional amino acid scoring pattern proposed by the World Health Organization of the United Nations (FAO/WHO 1973) classified the protein with 36% of the total residues as essential amino acids is ideal. These obtained results for the effect of water deficit on amino acid variations in the current research work are in agreement with the results obtained in the previous studies of Kovács et al. (2012) on wheat plants. Free radical scavenging causes osmotic adjustment due to the accumulation of amino acids (Keutgen and Pawelzik 2008). Proline is a compatible osmolyte which reduces water potential of cells and avoids toxicity of high ions concentration and a scavenger of reactive oxygen species (Verbruggen and Hermans 2008). Its accumulation can stabilize the structure of membranes and proteins to minimize the damage of cells under salt stress. Moreover, a large increase in proline precursors, glutamate and arginine was observed under water deficit stress. A part from the considerable accumulation of proline and its precursors in white lupine seeds also induced an increases in isoleucine (Ile) and leucine (Leu) contents. The interactive effect of different water deficit levels and foliar spraying of either melatonin or tryptophan on lupine plants had increased in total and essential amino acids content. The increase in amino acids (Table 6), especially proline, can be one of the causes of alleviating the inhibitory effect of water deficit by foliar applications of the tested materials.

Conclusion

Water deficit stress (75 and 50% WIR) significantly decreased growth, photosynthetic pigments, IAA and yield components. The decrease accompanied with significant increase in phenolic contents, hydrogen peroxide (H2O2), lipid peroxidation and some antioxidant enzymes. However, treatment of white lupine with different levels of melatonin and tryptophan significantly increased growth and yield attributes.

Application of 100 µM melatonin or 200 µM tryptophan were the most effective treatments, since they improved the productivity of white lupine in drought stressed and unstressed plants compared to the other treatments.

In future melatonin and tryptophan treatments could be used, in a large scale, as a safe and cheap method for increasing plant tolerance against water deficit stress conditions and thus, increase the plant productivity under different environmental conditions.

Abbreviations

WIR

Water irrigation requirements

MT

Melatonin

Try

Tryptophan

IAA

Indole acetic acid

CAT

Catalase

SOD

Superoxide dismutase

POX

Peroxidase

NR

Nitrate reductase

MDA

Malondialdehyde

TCH

Total carbohydrate

Funding

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

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

Contributor Information

Mervat Shamoon Sadak, Email: mervat_sh24@yahoo.com.

Amany Abd El-Mohsen Ramadan, Email: amanyramadan66@yahoo.com.

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