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Research Article
2026
:38;
12702025
doi:
10.25259/JKSUS_1270_2025

Supplementation of amino acids (L-Methionine and Proline) mitigates drought stress in canola (Brassica napus L.) plants through the modulation of physio-biochemical attributes and key antioxidant enzymes, and oxidative stress management

, , , , ,
aDepartment of Botany, Government College University, 38000, Faisalabad, Pakistan
bDepartment of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Lahore, Pakistan
cDepartment of Tropical Horticultural Crops of Hainan Province, School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, Haikou, China
dDepartment of Botany, MGDC Charar-I-Sharief-191112, Budgam, Jammu and Kashmir, India
eResearch and Development Cell, Lovely Professional University, Punjab-144411, India
fDepartment of Botany and Microbiology, King Saud University-11451, Riyadh, Saudi Arabia

* Corresponding author: E-mail address: nudrataauaf@yahoo.com (Nudrat Aisha Akram) and parvaizbot@yahoo.com (P Ahmad)

Received: , Accepted: ,
© 2026 Journal of King Saud University – Science - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Abiotic stress, as caused by a lack of water that inhibits a plant’s ability to meet its water requirements, results in stunted growth, wilting, early leaf drop, and compromised photosynthesis. A study was conducted to understand the role of amino acid-induced (L-methionine and proline) regulation in the growth and defense system of canola (Brassica napus L.) plants subjected to drought conditions. Drought stress [60% field capacity (F.C.)] considerably affected all physio-biochemical parameters. In both canola cultivars (cv. Punjab and cv. Rachna), drought stress enhanced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) enzymes, proline, glycine betaine, H2O2, MDA, and relative membrane permeability (RMP). Foliar-applied different treatments, i.e., control (no spray), Met, Pro, and Met + Pro of amino acids were given to canola cultivars. Of all treatments, Met increased the SOD enzyme activity, dry and fresh weights of roots, shoots, and root lengths, H2O2, siliquae dry weight, and the no. of siliquae. While exogenously applied, Pro enhanced the RMP, chlorophyll, total soluble proteins (TSP), and number of seeds per siliqua in cv. Punjab and MDA, ascorbic acid, activity of APX enzyme, siliqua fresh weight, and seed yield per plant in cv. Rachna. Met + Pro improved the dry and fresh weights, chlorophyll contents (a and b), leaf area, and POD enzyme activity in cv. Punjab and relative water content, proline, total phenolic, and activity of CAT enzyme in cv. Rachna. Overall, foliar applied Met + Pro showed more positive effects in improving the drought tolerance of canola plants in terms of growth, seed yield, and oxidative defense system of both canola cultivars, more promisingly in cv. Rachna.

Keywords

Antioxidants
Nitrogenous compounds
Oxidative defense system
Photosynthetic pigments
Water deficit conditions

1. Introduction

Scarcity of water is a significant barrier to crop development and productivity since it alters the fundamental physiological, molecular, and biochemical processes of vegetation in all phases of their growth (Hayat et al., 2023). Water scarcity is known to cause a myriad of issues in the plant’s typical operation, including a relative reduction in transpiration, photosynthetic rate, stomatal conductance, and water use efficiency (Kwon and Woo 2016; Cernusak 2020; Zhang et al., 2023) by generating multiple reactive oxygen species, thereby disrupting cellular membranes (Zhao et al., 2020). The synthesis of defense proteins, osmoprotectants, and antioxidants allows plants to withstand water-deficient circumstances by regulating water flux and causing cellular alterations (Hasegawa et al., 2000; Ashraf and Akram, 2009; Zhang et al., 2015).

The majority of plant-based amino acids are the building blocks of proteins. However, it is commonly recognized that stressed plants exhibit increased accumulation of certain amino acids. Methionine (Met), an amino acid that contains sulfur and is a member of the aspartate family, is necessary for a variety of functions in living things (Liao et al., 2022). For example, it has been reported that leafstalk abaxial epidermal thickness and width, keratinocytes, both dry and fresh weight of shoot, lengths, photosynthetic pigments, glycine betaine, total phenolics, and petiole abaxial epidermis thickness were improved by the supplementation of methionine to different plants (Akram et al., 2020; Mehak et al., 2021). Various methods such as mutagenesis, genetic engineering, and conventional breeding have been used to enhance Met content in several plants such as tobacco (Hacham et al., 2017; Oliveira et al., 2023), canola (Hacham et al., 2008), and Vicia faba (Hannoufa et al., 2014). One effective method for improving the growth performance and yield of cauliflower cultivated in drought-stressed environments is the foliar administration of proline and methionine (El-Bauome et al., 2022).

Proline is the most extensively distributed osmoprotectant in higher plants and is generally linked to osmotic adjustment under water stress (Hare and Cress 1997; Warsame et al., 2018; Nazir et al., 2024). Moreover, as a metabolic signal, proline has the ability to directly scavenge ROS and control antioxidant levels, which can help plant cells to withstand stressful cues (Liang and Shen 2018; Raza et al., 2023; Spormann et al., 2023). Exogenous proline administration improves gas exchange characteristics, for example, transpiration, stomatal conductance, and net carbon dioxide uptake in maize in a water-deficient environment (Szabados and Savoure 2010). In some other studies, exogenous application of proline reduced membrane permeability, ethylene, H2O2, and MDA contents, while it enhanced the chlorophyll accumulation and metabolites in creeping bentgrass (Ali 2007), barley (Ma et al., 2018), and kiwifruit (Abdelaal et al., 2020). Furthermore, in the proline-treated plants, the H2O2 level was much lower (by a factor of 2), and their MDA concentration was almost identical to that of perpetually irrigated plants after 4 days of recuperation (Abdelaal et al., 2020; Xia et al., 2020; Jurkoniene et al., 2023).

Like in most crops, canola growth and production are hampered due to drought stress (Raza et al., 2023). A considerable decline in numerous physio-biochemical traits, such as the quantity of seed oil and chlorophyll, the amount of non-enzymatic antioxidants, the activities of antioxidant enzymes, and their levels, is attributed to the growth impairment caused by the canola drought (Shafiq et al., 2014; Naheed et al., 2021). Although numerous studies have examined how exogenous Met or proline administration impacts how resistant various plants are to drought stress is available, the combined effect of Pro and Met on plant drought stress tolerance has been rarely researched. So, it was hypothesized that L-methionine and proline supplied exogenously may improve the drought tolerance of canola plants. So, the aim of the present study was to examine the underlying mechanisms of how exogenously applied Pro and Met impacted the drought tolerance capacity of the canola plant in terms of growth and different physio-biochemical traits by comparing the influence of applying two different amino acids together and individually.

2. Materials and Methods

The efficacy of two essential amino acids, proline (Pro) and L-methionine (Met), as well as the function of water scarcity regimes in regulating the physio-biochemical characteristics and growth of canola (Brassica napus L.) plants, were examined. The seeds of two canola cultivars, cv. Punjab and cv. Rachna were obtained from the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Four replications with a fully randomized design were used to set up this experiment. Sixty-four plastic pots were used, and 8 kg of sandy-loam (25%-75%) soil was placed in each container. In each pot, ten seeds were sown and given time to germinate. Six seedlings per pot were maintained after thinning. During the experimentation, the average day + night temperature, 25.4°C, average rainfall, 14.75 mm, soil moisture 21%, average relative humidity, 59 ± 2 percent, and daylight 7.5 h were noted. Based on the soil saturation percentage, the field capacity was measured and maintained throughout the experiment, which continued until the yield was harvested. Fifteen-day-old canola seedlings were subjected for 30 days to two irrigation regimes, control (100% F.C.) and drought stress (60% F.C.). These water stress levels were maintained based on soil moisture percentage and followed by the determination of field capacities. Field capacity was maintained on a daily basis in drought-stressed plants at the rate of 60% F.C. and control at 100% F.C. Various levels of Met 15, 30, and 30 mg L-1 (Akram et al., 2024), 20 mg L-1 (Mehak et al., 2021), and 10 and 20 mg L-1 (Akram et al., 2020) and Pro 20 mM and 40 mM, 40 mM (Ghafoor et al., 2019), and 20 mM have already been tested. On this basis, in this experiment, two doses, 25 mg L-1 of Met, and 40 mM (4605.2 mg L-1) of Pro were selected.

After 30 days of drought stress, four different treatments, i.e., control (no spray), Met (25 mg L-1), Pro (4605.2 mg L-1), and Met + Pro (25 mg L-1 + 4605.2 mg L-1) solutions were prepared in distilled water and foliarly applied to canola plants once a time during whole period of experiment while in control level (pots) simple distilled water was applied as a foliage spray. After 18 days of exogenous application of several treatments, the data were obtained for the following characteristics. For different physio-biochemical attributes, the 2nd or 3rd young leaf of each plant was harvested and stored in an ultra-low freezer at -20°C.

2.1 Shoot and root fresh and dry weights and lengths

From each replication, two plants of the same size were collected, separated into roots and shoots, and their fresh weights and lengths were recorded. Both the fresh biomass and the lengths of the roots or shoots were measured. The plant samples were sun-dried, then dried for 72 h at 60°C, and their dry weights were recorded.

2.2 Leaf area per plant

The leaf area per plant was calculated using the leaf length, leaf width, and leaf count.

2.3 Relative water content

A fully grown and expanded fresh leaf was taken from each plant, and its fresh mass (W1) was measured. Then, distilled water was used to dip the leaves for 3 h, and their turgid weights (W2) were measured. Following a period of drying at 65°C in an oven, the dry weights of these leaf samples were eventually calculated (W3). The relative water contents (%) were estimated using the formula below:

Relative   Water   Contents   ( % ) = W 1 − W 3 W 2 − W 3 × 100

2.4 Relative membrane permeability (RMP)

The relative membrane permeability (RMP) was determined by using the (Yang and Cortopassi 1998) procedure. The electrical conductivity (EC0) of a freshly ground 0.25 g leaf in 5 mL of demineralized water was measured after 2 h. Following an overnight placement of the samples, EC1 was ascertained. After 20 min of autoclaving at 120°C, the EC2 of the extracts was calculated using these samples. The RMP was calculated using the formula below.

RMP % = EC 1 − EC 0 / EC 2 − EC 0 × 1 00

2.5 Chlorophyll contents

Arnon et al (2024) was followed to find out the contents of Chl a and Chl b. Freshly obtained leaf tissue weighing 0.25 g was crushed in 80% acetone and stored overnight. After centrifugation, the OD at 663 and 645 nm from the supernatant was measured using a UV-visible spectrophotometer.

2.6 Proline content

Proline contents were calculated according to the description provided by Akram et al. (2024). Each 0.25 g fresh leaf was crushed in 5 mL of sulfosalicylic acid. So, the homogenized mixture was centrifuged 10,000 times for 10 min at 4°C at 10,000 × g. Next, 1 mL of each of the acid, ninhydrin, supernatant, and glacial acetic acid was added to a test tube. For an hour, these test tubes were heated to 95°C. After that, 2 mL of toluene was added to each test tube. After sufficiently shaking and cooling the mixtures in the test tubes, two layers formed; the top layer’s optical density (OD) at 520 nm was measured with a spectrophotometer.

2.7 Glycine betaine (GB)

For the determination of glycine betaine, (Grieve and Grattan 1983) was used. Each leaf sample, weighing 0.25 g was crushed in a pestle and mortar with 5 mL of distilled water. The mixture was centrifuged at 10,000g at 10°C for 10 min. Filtrate (0.5 mL) and 0.5 mL of 2N H2SO4 were added to a test tube, and 0.1 mL of KI3 was also added. The mixture was cooled for 90 min in an ice bath. Then, 3 mL of 1,2-dichloroethane was added, along with 1.4 mL of distilled water. After shaking the test tubes, two distinct layers formed, and the OD of the bottom liquid was measured at 365 nm.

2.8 Malondialdehyde (MDA)

A fresh 0.25 g leaf was homogenized in a pre-chilled pestle and mortar with 3 mL of 5% trichloroacetic acid (TCA). After that, the mixture was centrifuged for 10 min at 15,000g. Then, 0.5 mL of the supernatant was transferred into a test tube, and an aliquot part (2 mL) of 0.5% (w/v) TBA was added. The mixture was heated in a water bath for 50 min at 90°C, and cooled to room temperature. The OD of each treated sample was determined at 532 and 600 nm (Cakmak and Horst 1991).

2.9 Hydrogen peroxide (H2O2)

Using a chilled pestle and mortar, fresh leaf (0.5 g) was ground in 5 mL of 1% thiobarbituric acid. The supernatant was then extracted from the crushed substance by centrifuging it for 15 min at 12000g at 10°C. Then supernatant (0.5 mL) was mixed with 1M KI and 0.5 mL of potassium-phosphate buffer (pH 7.0). The OD was measured (Velikova et al., 2000) at 390 nm.

2.10 Ascorbic acid (AsA)

The AsA was determined by using (Mukherjee and Choudhuri 1983) method. In 10 mL of 6% TCA, a fresh 0.25 g leaf was crushed, 4 mL of the plant extract was added to each test tube, and an aliquot (2 mL) of 2% di-nitrophenyl hydrazine (prepared in 9N H2SO4) was added. After that, the test tubes were kept for 15 min at 95°C in a water bath. Following a period of cooling at ambient temperature, each test tube was filled with 5 mL of 80% (v/v) sulfuric acid. A spectrophotometer was used to measure the absorbance at 530 nm.

2.11 Total phenolics

Total phenolics were examined using (Julkunen-Tiitto 1985) method. The leaf sample was ground in 5 mL of 80% acetone. For ten minutes, at 10,000 × g, the extract was centrifuged. In a test tube, add 1 mL of Folin-Ciocalteu’s reagent, 2 mL of purified water, and 100 µL of supernatant, and then shake well. After that, 5 mL of a sodium carbonate solution (20%) was added to it. Distilled water was added to maintain the final volume to 10 mL, then the optical density of each treated sample was measured at 750 nm.

2.12 Total soluble proteins

A method (Bradford 1976) was applied to calculate the total soluble proteins. A fresh leaf (0.5 g) was crushed in 5 mL of phosphate buffer (pH 7.0) in a pestle mortar that had been refrigerated before handling. The samples were then centrifuged for 15 min at 12000g to extract the supernatant. A 100 µL sample of the supernatant was collected and transferred into a test tube, and 5 mL of Bradford reagent was also added. By using a spectrophotometer, the absorbance at 595 nm was measured.

2.13 Activities of superoxide dismutase (SOD), peroxide (POD), and catalase (CAT) enzymes

To determine the activity of the SOD enzyme, 50 µL enzyme extract (obtained from the extracts utilized to calculate the TSP) was contained in an Eppendorf tube, 250 µL potassium phosphate buffer, 100 µL Met, 400 µL distilled water, and 50 µL riboflavin were added. SOD was measured at 560 nm for the treated samples (Giannopolitis and Ries 1977).

The methodology of Giannopolitis and Ries 1977 was employed to determine the activity of the POD enzyme. The following were added: 50 µL of potassium phosphate buffer, 100 µL of hydrogen peroxide, 50 µL of enzyme extract, and 100 µL of 20 mM guaiacol. The absorbance was taken at 470 nm for 180 s using a spectrophotometer.

The CAT enzyme’s activity was measured using a procedure (Chance and Maehly 1955). Then, 5.9 mM KH2PO4 buffer (1.9 mL), H2O2 (1 mL), and enzyme extract (0.1 mL) were combined. The absorbance of all samples was measured at 240 nm for 3 min.

2.14 Yield attributes

After the crop was harvested, yield-related characteristics were noted. The number of seeds per siliqua, the number of siliquae per plant, the siliqua’s fresh and dry weights, and the number of seeds produced per plant were all recorded.

2.15 Statistical analysis

The analysis of variance (ANOVA) was executed on the data using Minitab 19 at the significance level (P < 0.05) after Tukey’s pairwise comparison test. Additionally, using R statistical software, data were provided for the multivariate analysis and correlation matrices.

3. Results

The current study found that both canola (Brassica napus L.) cultivars, Punjab and Rachna, leaf fresh and dry weights were considerably (P < 0.01) reduced by drought stress at 60% F.C. However, foliar treatment with L-methionine (Met), proline (Pro), and L-methionine + proline (Met + Pro) at the rate of 25 mg L-1, 4605.2 mg L-1, and 25 mg L-1 + 4605.2 mg L-1, respectively induced enhancement in both dry and fresh weights of both cultivars of canola (Table 1; Fig. 1). The data presented in Fig. 1 showed a substantial (P ≤ 0.01) improvement in these growth attributes by foliar-applied Pro as well as Pro + Met in both canola plants of both varieties in both conditions due to drought. Of both cultivars of canola, cv. Rachna was comparatively (P ≤ 0.01) healthier than cv. Punjab in shoot fresh and dry weights.

Table 1. Mean square values obtained from analysis of variance of data for different morpho-physiobiochemical characteristics of leaf of drought stressed canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline).
Source of variations df Shoot fresh weight Shoot dry weight Root fresh weight Root dry weight
Cultivars (Cvs) 1 183.7*** 0.238*** 0.030 ns 9.975ns
Drought (D) 1 140.6*** 0.830*** 0.748*** 0.054***
Treatments (T) 3 59.12** 0.079** 0.087*** 0.008*
Cvs x D 1 0.012ns 0.075* 0.001ns 0.008*
Cvs x T 3 5.142ns 0.010ns 0.004ns 8.921ns
D x T 3 10.96ns 0.005ns 0.040* 7.030ns
Cvs x D x T 3 9.784ns 8.511ns 0.011ns 8.202ns
Error 48 10.07 0.015 0.012 0.001
Source of variations Shoot length Root length Leaf area per plant Relative water content
Cultivars (Cvs) 1 220.0*** 18.02* 188.5*** 266.4**
Drought (D) 1 637.5*** 107.5*** 489.0*** 2676.7***
Treatments (T) 3 60.43*** 15.27* 79.97*** 338.0***
Cvs x D 1 0.694ns 2.262ns 4.917ns 16.17ns
Cvs x T 3 1.129ns 0.724ns 1.033ns 24.84ns
D x T 3 3.562ns 0.211ns 2.818ns 16.30ns
Cvs x D x T 3 0.833ns 0.156ns 5.284ns 19.40ns
Error 48 7.486 4.163 8.391 36.15
Source of variations RMP Chlorophyll a Chlorophyll b Chl. a/b ratio
Cultivars (Cvs) 1 104.6ns 0.101* 0.214** 0.493ns
Drought (D) 1 663.7*** 0.419*** 0.302*** 0.301ns
Treatments (T) 3 346.8*** 0.062* 0.043ns 0.364ns
Cvs x D 1 11.43ns 0.025ns 4.000ns 0.028ns
Cvs x T 3 10.20ns 0.004ns 0.005ns 0.413ns
D x T 3 13.07ns 0.003ns 0.002ns 0.082ns
Cvs x D x T 3 10.64ns 0.002ns 4.450ns 0.133ns
Error 48 32.50 0.017 0.021 0.479
Source of variations Total chlorophyll Proline GB H2O2
Cultivars (Cvs) 1 0.611** 0.775* 0.610ns 3373.6*
Drought (D) 1 1.433*** 1.434** 1.403ns 9152.1***
Treatments (T) 3 0.194* 1.049*** 18.11*** 3241.9**
Cvs x D 1 0.032ns 0.225ns 8.800ns 3620.0*
Cvs x T 3 0.003ns 0.141ns 1.579* 248.7ns
D x T 3 0.006ns 0.069ns 3.286*** 465.1ns
Cvs x D x T 3 0.003ns 0.073ns 0.941ns 309.1ns
Error 48 0.050 0.148 0.380 514.2
Source of variations MDA Total phenolics Ascorbic acid Superoxide dismutase
Cultivars (Cvs) 1 15527.9 *** 9.896ns 5.712ns 5.255ns
Drought (D) 1 10470.3*** 328.3*** 67.59*** 42.98***
Treatments (T) 3 2154.4*** 106.1** 11.17* 5.335*
Cvs x D 1 8.222ns 68.26ns 6.330ns 0.593ns
Cvs x T 3 191.0ns 15.18ns 4.436ns 0.883ns
D x T 3 1085.1* 1.969ns 1.654ns 1.338ns
Cvs x D x T 3 418.6ns 1.816ns 1.160ns 3.308ns
Error 48 287.3 17.99 3.625 1.691
Source of variations Catalase Peroxidase Total soluble proteins Ascorbate peroxidase
Cultivars (Cvs) 1 0.931*** 0.080ns 5.504* 13.65ns
Drought (D) 1 0.816*** 94.99** 62.27*** 558.8***
Treatments (T) 3 0.079* 67.46*** 6.482*** 65.26***
Cvs x D 1 0.068ns 16.18ns 3.055ns 13.26ns
Cvs x T 3 0.828ns 13.59ns 0.753ns 13.08ns
D x T 3 0.010ns 4.111ns 0.506ns 14.91ns
Cvs x D x T 3 0.004ns 25.81* 0.106ns 3.198ns
Error 48 0.021 8.970 0.872 9.558
Source of variations df Pod fresh weight Pod dry weight No of seeds per pod Seed yield per plant
Cultivars (Cvs) 1 0.090ns 0.134*** 0.765ns 112.8ns
Drought (D) 1 1.675*** 0.008ns 102.5*** 7431.8***
Treatments (T) 3 0.166* 0.009ns 34.68*** 1644.2***
Cvs x D 1 0.029ns 0.185*** 4.515ns 753.9ns
Cvs x T 3 0.042ns 0.007ns 1.515ns 29.84ns
D x T 3 0.025ns 0.002ns 3.932ns 641.4ns
Cvs x D x T 3 0.038ns 0.003ns 2.098ns 43.20ns
Error 48 0.046 0.009 2.515 246.5
Source of variations df No of pods per plant
Cultivars (Cvs) 1 32.11***
Drought (D) 1 21.77***
Treatments (T) 3 559.2***
Cvs x D 1 7.111*
Cvs x T 3 7.222***
D x T 3 9.629***
Cvs x D x T 3 16.29***
Error 48 1.027

significant; *, ** and *** = significant at 0.05, 0.01 and 0.001 levels, respectively; ns=nonsignificant.

Shoot and root fresh and dry weights (a-d), shoot and root lengths and leaf area (e-g) per plant of canola (Brassica napus) plants foliarly-treated with amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline; , S.E: Standard error.
Fig. 1.
Shoot and root fresh and dry weights (a-d), shoot and root lengths and leaf area (e-g) per plant of canola (Brassica napus) plants foliarly-treated with amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline; , S.E: Standard error.

Water deficiency caused a significant decrease in the root fresh and dry weights of canola plants. All foliar-applied treatments of amino acids increased both fresh and dry root weights, and of them, Met was more effective in promoting the canola plants’ fresh and dry root weights, particularly under a water-deficient environment (60% F.C.). According to our results, cv. Rachna was better than cv. Punjab in these growth characteristics in both water regimes (Table 1; Fig. 1).

Shoot or root lengths of both canola cultivars were reduced remarkably (P ≤ 0.05 and 0.001) due to water stress (Fig. 1; Table 1). Foliar-applied amino acids. individually or in combination, demonstrated the promising impact (P ≤ 0.001) of Met, particularly on cv. Punjab and Met + Pro in terms of shoot and root lengths under control (100% F.C.) and drought stress (Table 1; Fig. 1).

The data presented in Table 1 demonstrated that leaf area/plant was (P ≤ 0.01) affected by water shortage. However, amino acid exogenous administration proved effective, and Met + Pro treatment increased the leaf area per plant in cv. Punjab more significantly (P ≤ 0.001), while Met and Met + Pro had considerable effects on cv. Rachna at 60% F.C. (Fig. 1).

Under stress conditions, RWC was decreased considerably (P ≤ 0.001) in both cultivars of canola. Of all foliar treatments, Pro showed better results under both control and water deficit conditions, particularly in cv. Rachna, but Met + Pro had noticeable effects on RWC in the case of cv. Punjab in both stress or non-stress conditions (Table 1; Fig. 2).

Relative water contents (a) RMP, (b) and chlorophyll pigments, (a, b, a/b ratio and total) (c-f) of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.
Fig. 2.
Relative water contents (a) RMP, (b) and chlorophyll pigments, (a, b, a/b ratio and total) (c-f) of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.

Under water stress, the RMP was enhanced (P ≤ 0.001) at 60% field capacity. Exogenous treatments with amino acids were effective, and of all treatments, Pro performed better in both control and water-shortage conditions for both canola cultivars (Table 1; Fig. 2).

Water deficiency stress considerably (P ≤ 0.05) decreased chlorophyll content (a, b) and total chlorophyll pigments. Exogenous treatment of Pro markedly increased the Chl a and total Chl contents (P ≤ 0.05), particularly in cv. Punjab, in contrast to the other treatments (Table 1; Fig. 2). No considerable effect was observed between both canola cultivars in chlorophyll b contents. Drought stress (60% F.C.) and the foliar application of the amino acids, independently or in combination, cease changing the chlorophyll a/b ratio of both canola cultivars. Overall, at 60% field capacity, cv. Rachna performed better than the other canola cultivar (Table 1; Fig. 2).

Under water-deficient circumstances, there was a noteworthy (P ≤ 0.001) rise in the accretion of proline and glycine betaine (GB) contents, particularly in canola cv. Rachna (Fig. 3). Foliar-applied Pro was more beneficial (P ≤ 0.001) than Met in increasing proline buildup in addition to GB contents in canola plants (Table 1). Overall, cv. Rachna was more prominent in the buildup of proline and GB concentrations in conditions of water scarcity (60% F.C.).

Proline (a) glycine betaine, (b) H2O2 , (c) MDA, (d) total phenolics, (e) and ascorbic acid (f) (Mean ± S.E. n = 4) of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity). Met: L-Methionine; Pro: Proline.
Fig. 3.
Proline (a) glycine betaine, (b) H2O2 , (c) MDA, (d) total phenolics, (e) and ascorbic acid (f) (Mean ± S.E. n = 4) of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity). Met: L-Methionine; Pro: Proline.

Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations rose considerably in both canola cultivars (P ≤ 0.01 and 0.001, respectively) in water shortage conditions (60% F.C.). However, exogenously sprayed Met had a major effect in lowering the accumulation of MDA and H2O2. For both canola cultivars in water stress regimes, the contents of MDA and H2O2 were greater in cv. Punjab than cv. Rachna (Table 1; Fig. 3). 

Total phenolics increased while ascorbic acid (AsA) contents reduced considerably (P ≤ 0.01) under water stress. Although exogenous application, particularly Met + Pro, significantly (P ≤ 0.01) increased total phenolics in both canola cultivars (Table 1; Fig. 3). Although, in the case of AsA accumulation, the 100% and 60% F.C. water regimes were the most beneficial for exogenously administered Pro; both canola cultivars responded almost exactly to all exogenous treatments.

Under water-deficient conditions, both plants of canola cultivars, the enzyme activities of SOD, CAT, POD, and APX enhanced considerably. Exogenously applied amino acids were actual in boosting each of the four enzymes’ activity. Generally, Pro and Met + Pro were the most effective doses for promoting the activities of all enzymes (Table 1; Fig. 4). Both canola cultivars responded similar to these antioxidant enzymes’ activities under both control (100% F.C.) and 60% F.C. conditions (Table 1; Fig. 4).

Activities of (a) superoxide dismutase, (b) catalase, (c) peroxidase, and (d) ascorbate peroxidase enzymes and (e) total soluble proteins of canola (Brassica napus L.) plants foliarly-treated with amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.
Fig. 4.
Activities of (a) superoxide dismutase, (b) catalase, (c) peroxidase, and (d) ascorbate peroxidase enzymes and (e) total soluble proteins of canola (Brassica napus L.) plants foliarly-treated with amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.

In both canola cultivars, siliqua fresh and dry weights were dramatically reduced (P ≤ 0.05) when there was water scarcity. Foliar application of Pro, as well as Met + Pro, was most effective in increasing (P ≤ 0.001) the siliqua fresh and dry weights (Table 1; Fig. 5). For Siliqua dry and fresh weights, cv. Rachna outperformed the other canola cultivar.

(a-b) Pod fresh and dry weights, (c) no. of seeds per pod, (d) seed yield per plant, and (e) no. of pods per plant of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.
Fig. 5.
(a-b) Pod fresh and dry weights, (c) no. of seeds per pod, (d) seed yield per plant, and (e) no. of pods per plant of canola (Brassica napus L.) plants treated with foliar-applied amino acids (L-methionine and proline) subjected to control (100% field capacity) and drought stress (60% field capacity) (Mean ± S.E. n = 4). Met: L-Methionine; Pro: Proline.

Drought stress showed a substantial drop (P ≤ 0.001) in the number of siliquae per plant and yield/seed produced per plant of both canola cultivars. Regarding plant seed production, however, foliar application of Pro showed a substantial increasing effect (P < 0.001) and Met on a number of seeds per siliqua under water scarcity in both canola cultivars (Table 1; Fig. 5). In terms of these yield qualities, cv. Rachna excelled cv. Punjab.

4. Discussion

Under stress conditions, plant developmental processes are affected negatively, which eventually suppresses the yield and productivity of most crops (Rai et al., 2024). For example, it has been shown that drought stress negatively impacts a variety of molecular, biochemical, physiological, anatomical, and morphological processes necessary for plant growth and development (Chaudhry and Sidhu 2022). However, drought stress dramatically reduced the root and shoot fresh and dry weights of the canola cultivars Punjab and Rachna in the current study. Moreover, the seed yield-related data were similarly negatively impacted by drought stress in all canola plants. These findings are in accordance with earlier investigations that have shown drought-induced detrimental impacts on plant growth of several crops, especially maize (Wang et al., 2024), rice (Urmi et al., 2023; Karim et al., 2024), sunflower (Hanafy and Sadak 2023), and canola (Shafiq et al., 2014), which were ascribed to perturbance brought on by drought in various physio-biochemical processes.

In the current study, foliar-applied Pro and Met, applied individually or in combination, were significant for enhancing both dry and fresh weights of the roots and shoots of both cultivars of canola under stressful and non-stressful sets of conditions. Similar findings were also observed in proline-supplemented salt-stressed plants of maize (Irshad et al., 2024) and canola (El Habbasha et al., 2022), wherein a significant improvement in growth, concerning both fresh and dry weights of the shoot, was recorded because of proline supplementation. Likewise, Met application as an exogenous treatment was found to be more significant in enhancing root and shoot fresh and dry weights of different drought-stressed sunflower cultivars (Mehak et al., 2021). Moreover, a prominent role of Met was also observed by Akram et al., 2024 when foliar-applied 30 mg L-1 enhanced the weights of broccoli shoots under water stress.

Under severe water deficiency, one of the main reasons for decreased plant development and yield outcomes is elevated ROS generation. ROS interact with several vital intracellular constituents, including proteins, chlorophyll molecules, nucleic acids, and membrane lipids, to damage their ultra-structures and hamper their functions in a water shortage environment (Hasanuzzaman et al., 2021; Zulfiqar 2021). Plants have several defensive and coping strategies that are adaptable to the negative consequences of oxidative stress caused by drought. These include high non-enzymatic antioxidants levels, AsA, and total phenolic contents, together with high activities of enzymatic antioxidants like POD, CAT, and SOD, which can all work together to effectively lessen the harmful influences of drought on plants (Seleiman et al., 2021; Zulfiqar 2021). Usually, exogenously applied amino acids can perform as antioxidants and help plants to recover from the harmful effects of water stress (Teixeira et al., 2020). Amino acids are considered the most beneficial growth regulators that help plants to become resistant to various abiotic stresses (Ali et al., 2019). Methionine (Met) is a necessary amino acid that participates in different metabolic responses. It is crucial for the synthesis of methane and protein metabolism. Its methyl group, coupled to sulfur, acts as a precursor to activate S-adenosylmethionine (Lenhart et al., 2015). For example, high endogenous Met accumulation within the cells reduced the consequences of drought by boosting the amount of photosynthetic pigments and proline, although functions of antioxidant enzymes such as SOD, POX, and CAT (Merwad et al., 2018). Furthermore, it was discovered that exogenous proline administration was successful in enhancing the activity of important enzymatic antioxidants, including CAT, POX, and SOD, in salt-stressed maize (Pa et al., 2018) and sweet pepper (Abdelaal et al., 2019) plants. Also, Pro is effective in increasing the activities of enzymatic antioxidants in rice plants, including CAT, SOD, and POX, after the plants are stressed by dehydration (Hanif et al., 2021). Different plants subjected to salinity and drought conditions have shown considerable Met, Pro, and Pro + Met growth improvement in different plants due to supplementation of Met, Pro, and Pro + Met, such as cabbage (Haghighi et al., 2020), tomato (Almas 2021), sunflower (Mehak et al., 2021), cauliflower (El-Bauome et al., 2022), rapeseed (Moghadam et al., 2022), rice (Urmi et al., 2023), and maize (Shahid et al., 2023). In our study, both canola cultivars’ shoot and root lengths were significantly reduced when there was a 60% F.C. However, exogenously-applied Met + Pro remained more operative than their individual applications in promoting growth in the drought-stressed plants of both canola cultivars. In a previous study, interestingly, it was observed that exogenously administered Met improved the shoot and root lengths of drought-stressed maize crops (Shahid et al., 2023). Previously observed the outcomes in wheat plants under salinity stress (Hammad and Ali 2014).

RMP and relative water content (RWC) are potential indicators of plant drought tolerance (Akram et al., 2018; Hasanuzzaman et al., 2018; Sharif et al., 2018). Both plant attributes are adversely impacted by the conditions of water scarcity. As an illustration, exogenous spray of proline restored the leaf RWC and RMP of barley plants in water shortage circumstances (Abdelaal et al., 2020; Ibrahim et al., 2020). We analyzed that in a simulated drought with a continually increasing water deficit, exogenously supplemented proline had positive effects related to the relative water content of rapeseed leaves. However, the collective application of Pro and Met showed a synergistic effect in regulating RWC in both canola cultivars, being more promising in cv. Punjab during drought-stress environments.

Drought produces oxidative stress, which is believed to significantly reduce chlorophyll content and rate of photosynthesis because of the excessive production of ROS (Samanta et al., 2023; Avalbaev et al., 2024). In the present study, the concentrations of chl a, b, and total chlorophyll declined in both canola cultivars in water-deficient conditions; it is found to be the most common comeback of plants under drought stress (Norouzi et al., 2024; Zafar et al., 2024). However, exogenously applied Pro effectively improved the chlorophyll a and total chlorophyll in cv. Punjab. Some earlier published studies have also suggested that methionine, proline, and melatonin applied externally increased the amount of chl. in the leaves of many plants, including sunflower (Akram et al., 2020), and potato (Abd Elhady et al., 2021; El-Yazied et al., 2022). In addition, Shahid et al., 2023 and Zahedi et al., 2023 showed that varying levels of Met and Pro resulted in enhanced levels of chlorophyll pigments in maize and grape plants, respectively. It has been suggested that higher proline concentration controls the activity of chlorophyllase, thereby hampering the breakdown of chlorophyll and resulting in improved chlorophyll concentration (Elsheery and Cao 2008).

In the current investigation, GB and proline content rose in the drought-stressed plants of both canola cultivars. It has already been observed in several crop plants suffering from drought, such as bitter gourd (Akram et al., 2020), maize (Shahid et al., 2023), Brassica napus (Ali et al., 2023), and broccoli (Akram et al., 2024). According to Ashraf and Foolad 2007, a significant accretion of GB or proline in plants was usually known to be a measure of one’s ability to handle stress. (Kaya et al., 2013) suggested that an endogenous high GB level is essential for achieving enhanced plant stress tolerance, as it functions as a potential ROS scavenger under stressful conditions. Analogous to the results of the present study, in a few earlier investigations, reports stated that supplementation of Met and Pro is highly beneficial for increasing the accumulation of GB and proline in plants during stress (Zhang et al., 2009; Shahid et al., 2023; Sehar et al., 2023).

Drought-persuaded oxidative stress significantly increased the H2O2 concentration in plants, which can damage the cellular membranes (Niu and Liao 2016). In the present study, H2O2 and MDA concentrations were elevated in both cultivars of canola when subjected to water shortage conditions. Similar outcomes have previously been observed in plants of various crops, including sunflower (Mehak et al., 2021), maize (Shahid et al., 2023), tomato (Almas 2021), and cauliflower (El-Bauome et al., 2022) in response to water shortage conditions. However, exogenously applied Met significantly reduced the formation of MDA and H2O2 in both canola cultivars during situations of water starvation. However, foliar applied Pro (1.0%) reduced drought-driven overproduction of H2O2 and MDA in wheat plants under stress conditions (Parveen et al., 2024).

Due to its antioxidative potential, ascorbic acid (AsA) helps regulate plants’ aerobic metabolic activities and photosynthesis, which would otherwise be affected by oxidative stress (Celi et al., 2023). In the current study, both canola cultivars showed a rise in AsA and total phenolics in a condition with low water. The foliar application of Met + Pro enhanced total phenolics in both canola cultivars, but exogenously applied Pro (40 mM) improved the levels of AsA in the canola plants under both water regimes (60% F.C. and 100% F.C.). Methionine application was also shown to enhance the leaf total phenolics in maize (Shahid et al., 2023). In contrast, in another study on sunflower, no noticeable variation was ascertained in total phenolics and AsA due to exogenously applied 20 mg L-1 Met under water shortage conditions (Mehak et al., 2021). However, exogenously applied proline significantly enhanced the proline as well as AsA in plants of quinoa (Yaqoob et al., 2019) and canola (Sakr et al., 2012).

In the current study, enzymatic antioxidant activities such as catalase, SOD, POD, and ascorbate peroxidase (APX) enhanced in plants of both canola cultivars during water stress. Generally, external spray of Pro and Met + Pro were the most effective doses for boosting both canola cultivars’ antioxidant enzyme activities under water-limited regimes. It has been recently shown that foliar-applied amino acids (arginine, methionine, glutamine, and proline) were efficient in raising the SOD, POD, CAT, and APX activities in a variety of plants, including marigold (Haghparvar et al., 2023), peanut (Wang et al., 2024), and sugarcane (Jacomassi et al., 2024), thereby improving the oxidative defense potential of plants. Foliar-applied Met has been shown to significantly improve the activities of POD, CAT, and SOD enzymes in broccoli both during stress and non-stress situations (Akram et al., 2024). Exogenously applied proline (30 mM and 10 mM) in the salt-stressed plants of maize (Pa et al., 2018) and sweet pepper (Abdelaal et al., 2019) enhanced the activities of SOD, POX, and CAT, respectively. But in drought-stressed rice (Hanif et al., 2021) and chickpea plants (El-Beltagi et al., 2020), 30 mM Pro was more functional in enhancing the CAT, POX, and SOD activities.

5. Conclusions

In conclusion, water-deficit stress considerably decreased the shoot and root fresh and dry weights, shoot and root lengths, chlorophyll pigment, leaf area, relative water contents, total soluble proteins, siliqua fresh and dry weights, number of seeds per siliqua, seed yield per plant, and number of siliquae per plant. An increased RMP, proline, glycine betaine, H2O2, MDA, total phenolics, and the activities of SOD, CAT, POD, and APX in both canola cultivars were observed. Application of amino acids (L-methionine and proline) induced a significant improvement in plant growth and the defense system of canola. Overall, foliar applied Met + Pro showed a more effective response in enhancing the drought stress tolerance in terms of growth, yield, and oxidative defense system of both canola cultivars.

Acknowledgment

The authors extend their appreciation to the Ongoing Research Funding Program, (ORF-2025-686), King Saud University, Riyadh, Saudi Arabia

CRediT authorship contribution statement

Saliha Umer and Nudrat Aisha Akram: Concept, design, the definition of intellectual content, experimental studies, data acquisition, data analysis, statistical analysis, manuscript preparation, Muhammad Ashraf: Literature search, data acquisition, manuscript editing, and review, Muhammad Ahsan Altaf: Data acquisition, data analysis, and manuscript preparation, Parvaiz Ahmad: Concept, design, Statistical analysis, manuscript preparation, and manuscript editing, and review, Abdel-Rhman Z Gaafar: Data analysis, literature search, manuscript preparation.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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