Interactive effects of melatonin and salicylic acid on Brassica napus under drought condition

Aims It is imperative to understand the synergetic actions of Melatonin (MET) and salicylic acid (SA) in counteracting drought stress. A pot experiment was performed to investigate the interactive effects of exogenously applied MET and SA on morphology, physiology, and biochemical characteristics of drought-stressed canola ( Brassica napus ). Methods Two cultivars (Super canola and Faisal canola) were used. MET (0.1 µM) and SA (0.50 mM) were applied as seed priming and foliar spray. Plants were exposed to severe drought (45% field capacity) at the reproductive stage. Results Drought stress caused a significant reduction in shoot length (19%), plant fresh (15%) and dry (17%) biomass, yield (30%), photosynthetic rate (43%), water potential (29%) and osmotic potential (33%) values. Increased activity of free amino acids (20%), total soluble sugars (5%), and different antioxidant enzymes like catalase (27%), peroxidase (23%), superoxide dismutase (20%) and ascorbate peroxidase (35%), were recorded under drought. Seed priming and foliar application of MET and SA reduced the negativity of drought. Priming of seeds with MET, as well as synergetic application of MET priming and SA foliar spray, were the most effective treatments, showing the most significant growth criteria (22% increase in plant height, 46% in fresh and 40% in dry biomass), yield components (49%), water (54%) and osmotic potential (28%), and endogenous MET (17%) and SA (16%) content. Super canola cultivar showed better growth than Faisal canola. Conclusion Study showed that the synergetic application of MET and SA has the potential to induce stress resistance and enhance canola plants' growth under drought.


Introduction
As the world population which is currently about 7 billion is expected to reach 9 billion by 2050, food security to support this much population is threatened to a larger extent by different types of Brassica crops commonly grown as oilseed crops in Pakistan include Rapeseed (B. rapa and B. napus) and mustard (B. juncea). Among these, mustard is traditionally used in Pakistan, however, its oil is not preferred in the preparation of vegetable oil or ghee due to the presence of very high amounts (40-70%) of erucic acid in oil, which is not suitable for human consumption (Onemli 2014). The annual edible oil requirement of Pakistan is about 1.95 million tonnes, of which only 18% is obtained from the local resources and the rest 88% is imported (Mustafa et al. 2017). B. napus, being indigenous species, has all the potentials to overcome the problems of low local oil production. It contains 44-46% of good quality oil, with its meal having 38-40% of protein contents rich in lysine, methionine, and cystine amino acids (Onemli 2014).
The yield of crops in most areas of Pakistan is drastically affected by drought. Canola is cultivated in high rainfall regions and shows low production in areas with low rainfall ( Present research work aimed to evaluate the response of two B. napus cultivars to drought stress concerning their growth, yield, and biochemical responses. In particular, our goal was to investigate the potential of the combined application of melatonin and salicylic acid, via pre-sowing seed treatment and foliar spray, in inducing drought resistance and mitigating the drought stress effects on two canola cultivars.

Experimental setup
To check the e cacy of melatonin (MET) and salicylic acid (SA), potential plant growth regulators, in reducing the adversity of drought stress on the growth of canola (Brassica napus L.), exogenous application of these compatible solutes as seed priming, and foliar spray methods were employed in soil culture pot experiment. An experimental area of the department of Botany, PMAS Arid Agriculture University, Rawalpindi was used for the experiment. Two canola cultivars, namely Super canola (V1) and Faisal canola (V2) were used. Seeds were attained from National Agriculture Research Center Islamabad, Pakistan.
Before sowing, seeds were surface sterilized with 10% Clorox. Potassium, Phosphorous, and nitrogen fertilizers were applied to the soil prior to seed sowing. Soil analysis was also carried out before starting the experiment. Two plant growth regulators, Melatonin (Sigma-Aldrich, Lot# SLBZ6359) and Salicylic acid (Sigma-Aldrich, Lot# STBH3314) were used in present study. MET (0.1µM) and SA (0.5mM) were applied in two different ways, rstly, seed priming was done for 24 hours using 0.1µM concentration of MET, 0.5mM concentration of SA, and a combined seed priming treatment of 0.1µM MET and 0.5mM SA, later foliar spray of MET, SA and MET plus SA combined was used at adult growth stages in both control and water stressed plants. 4 hours seed soaking in distilled water (for control) was also done, before sowing. Drought stress was established at 45% eld capacity 60 days after sowing, and the plants were grown in the 2020-2021 growing season. The treatments application included controlled irrigation (T 0 ), MET priming (T 1 ), MET foliar spray (T 2 ), SA priming (T 3 ), SA foliar spray (T 4 ), MET plus SA combined priming (T 5 ), MET plus SA combined foliar spray (T 6 ), MET priming combined with SA foliar spray (T 7 ), SA priming combined with MET foliar spray (T 8 ). 10 days after the imposition of drought, data for different photosynthetic attributes of each plant was recorded by using portable infrared gas analyzer (IRGA). Leaf samples were then collected for further physiological and biochemical analysis.

Morphological and yield attributes
Data for different morphological parameters, i.e., Plant height (cm), root length (cm), plant fresh and dry biomass (g), and yield attributes i.e., No. of siliqua per plant, No. of seeds per siliqua was recorded.

Gas exchange parameters
Infrared gas analyzer (LCA-4 ADC) was used for the estimation of gas exchange characteristics. These measurements were carried out from 10:00 to 14.00 hours. Measurement of photosynthetic rate, transpiration rate, water use e ciency and stomatal conductance were made using the young leaf. Bates et al. (1973) method was followed for estimation of protein contents. 0.2g of fresh leaf material were mixed with 10ml of phosphate buffer. 0.5ml of this extract was then added with 3ml of bio-red dye and 0.5ml of distilled water. Sample absorbance was checked at 595nm. Total amino acids were estimated according to the methodology given by Sohail et al. (2018). 0.2g of fresh leaf material was mixed with 10ml of phosphate buffer. 1ml of this extract was then added with 1ml of ninhydrin and 1ml of pyridine solution. Obtained mixture was then boiled at 100 ºC for 30 mins and sample absorbance was checked at 570nm by using Cecil 2021 Photo spectrometry. Dubois et al. (1956) methodology was used for this purpose. 0.2g of fresh leaf material was mixed with 10ml of 80% ethanol. After ltration, 0.5ml of ltrate was mixed with 0.5ml of distilled water and 1ml of 18% phenol solution. After keeping the mixture as such for an hour, 2.5ml of sulphuric acid was added. Absorbance was noted at 490nm.

Antioxidant Enzymes
Fresh leaf material was grounded in liquid nitrogen, crushed with pestle mortar, and powdered material was stored at -80 0 C for further use. Methodology given by Aebi (1984)

Morphological Attributes
Drought stress affected different morphological attributes of plants, and 19 percent reduction was recorded in shoot lengths of both canola cultivars as compared to control ( Fig. 1-a). The application of MET and SA caused a signi cant increase in plant height in both normal and stressed conditions. MET priming appeared to be most effective treatment of all under both normal and drought-stressed conditions, as an increase of 22% in plant height was recorded in MET primed (T 1 ), super canola plants compared with non-treated drought-stressed plants ( Fig. 1-a). Similarly, plant height was improved by 17% in plants treated with MET priming combined with foliar spray of SA (T 7 ). Shoot fresh and dry biomass also show a declining trend when plants were exposed to drought and a reduction of about 14% in fresh biomass and 17% in dry weight was recorded ( Fig. 1-c, d). Application of plant growth regulators proved bene cial in reducing the negative effects of drought shoot fresh and dry biomass. ME priming showed better results among all treatments where about 46% better fresh biomass and 40% better dry biomass was recorded in both canola cultivars under drought stress ( Fig. 1-c, d). These results were statistically non-signi cant from the plants treated with combined application of melatonin priming and salicylic acid foliar spray. Overall, V1 showed better growth under drought than V2.

Photosynthetic Attributes
Photosynthetic rate showed a signi cant reduction (43%) after canola cultivars were exposed to drought.
Seed priming treatments with plant growth regulators improved the photosynthetic rate of both cultivars under normal as well as stressed conditions (Fig. 2). Seed priming treatment with MET (T1) caused a signi cant increase of 43% in photosynthetic rate, followed by those plants which were treated with combined ME and SA (T5) priming ( Fig. 2-a). A similar sort of drought stress caused reduction in transpiration rate (73%) and stomatal conductance (55%) was recorded in both canola cultivars ( Fig. 2-b, c). Seed priming treatment with MET (T1) and SA (T3) showed better results and improved transpiration rates and stomatal conductance under drought in both canola cultivars ( Fig. 2-b, c). Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved all photosynthetic attributes in stressed environment, but their impact was not as signi cant as of all other treatments involved. Among the cultivars, V1 showed better results than V2.

Water Relations
Declining trend was recorded in water and osmotic potential values of canola plants after exposure to drought stress ( Fig. 3-a, b). Seed priming and foliar spray application of both plant growth regulators improved water potential and osmotic potential under both controlled and stressed conditions. Maximum water and osmotic potential values under normal and drought-stressed environments were recorded in MET primed plants (T1), followed by combined treatment of MET priming and SA foliar spray (T7). Combined foliar spray treatment (T6) of both growth regulators didn't show as signi cant effect on these parameters as shown by other treatments involved ( Fig. 3-a, b).

Bio-Chemical Attributes
An increase in compatible solutes concentration i.e., soluble proteins, free amino acids, and total soluble sugars were recorded after drought stress treatment in canola plants (Fig. 4). An increase of 15% was recorded in protein contents under drought stress, compared to water-treated plants. These Proteins contents were further increased by MET and SA application, and the maximum increase was recorded in MET primed (T1) plants ( Fig. 4-a), where an increase of 25% in protein contents was recorded under drought, compared to non-treated drought-stressed plants (T0). Similarly, about 20% more free aminoacid contents were recorded in drought stress, as plants accumulate more amino acids to cope with drought stress. Amino-acid contents were further enhanced by seed priming treatment of MET combined with foliar spray of SA (T7), where about 34% more amino-acid activity was found compared with nontreated (T0) water-stressed plants ( Fig. 4-b). A slight increase (5%) was recorded in total soluble sugar contents of drought-stressed cultivars, compared to well-watered plants. Hormonal priming treatment further enhanced these contents, and MET priming treatment combined with SA foliar spray (T7) proved to be most effective under drought stress. Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved all biochemical attributes under water de cit conditions, but their impact was not as signi cant as of all other treatments involved ( Fig. 4-c). Among the cultivars, V1 showed better biochemical activity than V2.

Antioxidant activity
Antioxidant levels i.e., Peroxidase (POD), Superoxide Dismutase (SOD), and catalase (CAT) activity increased in drought exposed canola plants, compared to well-watered plants (Fig. 5). An increase of 23% in POD levels was observed in drought-stressed canola cultivars. Application of plant growth regulators further enhanced POD contents under both normal and stressed conditions. Maximum results were shown by combined treatment of MET as seed priming and SA as foliar spray (T7) under both normal and drought stress conditions ( Fig. 5-a). These results were statistically non-signi cant from MET priming treated plants (T1). Similarly, an increase of 20% in SOD contents was recorded in droughtstressed canola plants. MET priming combined with SA foliar spray (T7) further enhanced these contents and about 17% increase under normal and 28% increase under stressed conditions was recorded ( Fig. 5b). CAT contents also showed a signi cant increase (27%) after drought exposure (Fig. 5-c). MET priming (T1) further increased catalase contents by 35% under stressed environment and by 14% under normal conditions. Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved SOD, POD and CAT levels in stressed environment, but their impact was not as signi cant as of all other treatments involved ( Fig. 5-a, b, c). Among the cultivars, V1 showed better antioxidant activity than V2.

Yield Attributes
Yield attributes of canola showed signi cant reduction after exposure to drought stress, where about 30% less siliqua per plant and 18% and 30% lower number of grains per siliqua were recorded in Super and Faisal canola respectively, as compared to control ( Fig. 6-a, b). However, a considerable increase (49 and 47%) in siliqua per plant and an increase of 40 and 56% in the number of grains per siliqua was recorded in Super (V1) and Faisal canola (V2) respectively, after treating plants with MET and SA under drought.
Maximum no. of siliqua per plant under drought conditions were shown by MET priming treated plants (T1), followed by plants treated with SA (T3) priming ( Fig. 6-a). Similarly, the maximum number of grains per siliqua was recorded in those plants which were treated with MET priming combined with foliar spray of SA (T7) in a stressed environment (Fig. 6-b). Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved plant yield attributes in both controlled and stressed environment, but their impact was not as signi cant as of all other treatments involved. Among the cultivars, V1 showed a slightly better yield than V2.

Relationship and multivariate analysis
In order to verify the relationship between different studied attributes, we have plotted Pearson correlation and principal component analysis (PCA-biplot) ( Fig. 7-8). This analysis disclosed correlation (positive and negative) among various parameters of canola cultivars under drought and normal conditions. It has been noticed that drought stress imposed signi cant impact on all the parameters. Fig. 7(a-b) showed signi cantly positive correlations among biochemical attributes, enzymatic antioxidant, and plant water relations both under drought and control conditions. On the other side, biochemical parameters showed a negative correlation with root length and stomatal conductance. Plant height and fresh weights were positively impacted by water relation attributes, but transpiration rate showed negative relationship with them. Overall transpiration rate (E) had negatively correlation with all other attributes under control conditions but showed almost zero relationship with number of grain per silique and number of seed per plant, while photosynthetic rate had shown positive correlation both under drought as well as controlled conditions due to application of melatonin and salicylic acid (Fig. 7a, b).
Our Pearson's correlation among various attributes of both canola varieties have been further validated by PCA-Biplot. Fig. 8 represents PCA-Biplot of different attributes in canola plants subjected to drought stress with foliar and primed seed treatments of growth regulators. Principal component analysis executed the degree of association between variables under drought and controlled environment. Both dimensions of PCA i.e., Dim-1 and Dim-2 jointly elaborated 82.6% variability in the dataset. They showed obvious and marked separation of attributes under stressed and non-stressed conditions. Dim-1 showed 44.9%, while Dim-2 observed 37.7% variation to the total variance. There was a clear separation in studied parameters under normal and drought stress. Biochemical and antioxidant traits showed more relationship and impact under drought while growth attributes showed better relation in controlled conditions (Fig. 8).

Discussion
Keeping in view the drastic effect of drought stress on plant growth, the present study was conducted to check out the e cacy of plant growth regulators (MET and SA) in enhancing morphological, biochemical, and physiological attributes of canola cultivars grown under drought stress conditions. The role of hormones in enhancing plant growth under stressed environments is a well-documented phenomenon.
Water stress is the major limiting factor responsible for the reduced growth and yield of crops (Sattar et al. 2021). Results obtained from our study showed all the growth attributes i.e., shoot and root length, plant fresh and dry biomass, as well as yield parameters, showed signi cant reduction after exposure to water stress in both canola cultivars. These results are Present research work showed a signi cant reduction in net photosynthetic rate and stomatal conductance in both canola cultivars after exposure to drought stress. Seed priming with melatonin and salicylic acid increased photosynthetic rate and stomatal conductance, both under control and stressed conditions. Leaf relative water content was also signi cantly increased by hormonal treatment in a stressed environment. Foliar spray of salicylic acid in combination with melatonin priming also showed statistically signi cant results, signifying the synergetic effect of these growth regulators on photosynthetic attributes under stressed conditions. Our results are in line with those obtained by Cui et al. (2017), who observed similar sort of melatonin effects on photosynthetic rate and stomatal conductance while working on stress-exposed wheat plants.

Conclusion
Present research work has proved that exogenous application of melatonin and salicylic acid has the potential to induce drought tolerance in canola plants, though melatonin appeared to be much more effective than salicylic acid. The combined application of melatonin priming, and salicylic acid foliar spray showed a signi cant effect on plant growth and can be very effective in inducing drought resistance in canola cultivars. Among the cultivars, Super canola (V1) showed much better growth and yield than Faisal canola (V2), so Super canola can be a better choice in drought affected areas and can be used for future studies.

Declarations
Con ict of interest The authors declare no con ict of interest.   Effect of different treatments of priming and foliar spray of Melatonin and Salicylic acid on POD activity (a), SOD activity (b) and Catalase activity (c) of two canola cultivars under normal and drought stress.
Small letter above each bar shows statistical differences at p <0.05 Page 22/24