Exogenously Applied Silicon and Zinc Mitigates Salt Stress by Improving Leaf Pigments and Antioxidant Activities in Canola Cultivars

Exogenous application of zinc (Zn) and silicon (Si) might serve as potent strategy to boost productivity of oil seed crops including canola in a saline environment. A trial was conducted with an aim to determine the phenotypic divergence among canola cultivars (Sandal and Rachna) under varying doses of Zn and Si (35 ppm and 70 ppm) applied solely and in conjunction with each other under two level of salinity stress (SS). Different morphological and physiological traits of canola cultivars were taken as response variables. The results revealed that SS adversely affected the leaf pigment and shoot length along with their fresh and dry weights, while antioxidant activities were increased especially under exogenous application of Si + Zn = 70 ppm. Under salinity stress conditions the root length and their fresh as well as dry weight increase as compared to the controlled plant which are grown under normal conditions without salinity stress. Moreover, canola cv. Sandal outperformed in terms for shoot–root length and their fresh and dry weight as well as the leaf pigments contents. The co-application of Si + Zn = 70 ppm exhibited the highest shoot–root length (17.64 cm—16.47 cm) and their fresh (2.60 g—1.89 g) as well as dry weight (0.73 g—0.29 g). The same treatment combination resulted in the maximum leaf pigments such as chlorophyll a (6.63 g/mg FW), chlorophyll b (4.37 g/mg FW) and total carotenoids content (2.38 g/mg FW).


Introduction
Worldwide, oil seed crops are imperative for human consumption as compared to wheat rice, oat, barley and sugar crops by offer 2.5 times higher calories. Among seeds crops, canola (Brassica napus) belonging to family Brassicaceae is the most significant oilseed crop globally [1][2][3] as its seed contains 45% oil and 25-35% protein [4,5]. Additionally, canola can be successfully cultivated in new land to avoid competition with different crops inhabiting the old cultivated lands [6][7][8]. Its oil is utilized in cooking, bioprocess, and the organic agrochemical industries with the grain used as protein rich animal feed after oil exploration. However, canola is sensitive to salt stress (SS) which seriously hampers its growth and reduces yield [9][10][11]. The SS alters the morphology and disrupts vital physiological processes of crop plants [9,11,12]. The ionic imbalance, changed vapor pressure, reduced nutrient absorption and photosynthetic rate are caused by SS [13][14][15][16]. In addition, SS inhibits seed germination and root propagation of canola which leads to reduced uptake of water and nutrient [13]. Moreover, SS impairs vegetative growth (number of leaves, leaf area, number of branches etc.) and reproductive phases (grain filling, number and length of pods, grain number and weight etc.) of canola [14,15].
For mitigating the adverse impacts of salinity on growth and development of canola, exogenous application of silicon (Si) can be a biologically viable option [10,16]. In irrigation water at a certain level (0.1-0.6 mm), Si is available to plants as mono silicate [Si (OH) 4 ]. The exogenous application of Si is attracting the attention of researchers due to its important functions in plant physiology, nutrition, and defense response plants grown in saline environment. Previously, Si application improved resilience against SS by producing larger seeds [17][18][19] and altering the leaf morphology which promoted plant development of winter canola [11,[20][21][22]. The application of Si increased the volume, size, and weight of roots, reducing the impact of water scarcity on crop plants by allowing them to absorb more water and nutrients [2,17,18]. However, dose optimization of Si for foliar spray in order to mitigate the adverse effects of salinity continues to remain an explored aspect, while contradictory findings have been reported previously.
Besides Si, zinc (Zn) is another micronutrient that is radially absorbed by roots and transported as a divalent cofactor via the symplastic and apoplastic pathways [23]. Many key enzymes, such as antioxidant enzyme, alcohol dehydrogenase and RNA molecule use it as a building element and coenzyme. It also helps plants with photosynthetic rate, pollen generation, carbohydrate and protein metabolism, nutrient uptake, and seed quality [20,24]. The exogenously applied Zn boosts protein synthesis and oil content in oil seed crops [4,21,22]. Additionally, Zn plays a direct role in the manufacturing of growth factors like dopamine, which leads to the production of more plant cells and dry matter [25]. Moreover, Zn also plays a crucial function in protein structure in plants as a co-factor for at least 300 enzyme systems [26] and protects plant against reactive oxygen species (ROS) by enhancing the antioxidant system [27,28]. Positive impacts of Zn on production, oil content, and FA concentration of field-grown canola include enhanced rate of photosynthesis and translocation of photo-assimilate, increased frequency of hydroxylase glycolytic pathway and ribulose diphosphate carboxylase and oxygenase, and changes in nucleic acid and protein biosynthetic pathways [4,21,22]. Foliar Zn treatment had a considerable influence on seed production, energy content, lipid content, and metabolic functions of canola [29]. Compared to sole application, the co-application of Si and Zn boosted plant height, number and dry weight of stem, leaf chlorophyll content, relative water content, stomatal conductance, and photosynthetic rate [30].
However, research findings are scant regarding comparative efficacy of Si and Zn doses applied in conjunction with each other for canola genotypes grown in saline environment. There is dire need to evaluate genetically divergent canola cultivars response to Si and Zn applied solely and in co-application for alleviating the deleterious effects of salinity. Thus, it was hypothesized that foliar applied Si and Zn might alleviate the adverse impacts of salinity by boosting morphological traits and improving physiological functioning of the canola plants under varying levels of induced salinity. Hence, the present study was designed to examine the role of foliar applied Si and Zn on physiological mechanisms of canola seedlings under normal and salt stress condition. The ultimate aim was to sort out the most performing dose of Zn and/Si for salinity mitigation along with finding out the superior cultivar of canola based on morphological divergence under induced salinity.

Experimental Detail
Salt stress experiment at the vegetative growth stage was conducted in the wire-house located at Department of Botany, University of Central Punjab, Constituent College Yazman Road Bahawalpur in October 2021, under normal and salt stressed conditions. Seeds of two spring canola varieties (Sandal & Rachna) were procured from Oil research Institute located at Ayyub Agriculture Research Institute (AARI) Faisalabad, Pakistan. This trial was conducted using a completely randomized design (CRD) with a factorial arrangement having three replications. The experiment was comprised of two canola varieties (V 1 = Sandal and V 2 = Rachna) and foliar sprays including control, Si = 35 ppm, Si = 70 ppm, Zn = 35 ppm, Zn = 70 ppm, Si + Zn = 35 ppm and Si + Zn = 70 ppm. The Zn and Si doses were applied as foliar sprays at three leaf stage, while their sources were Zn sulfate (35% Zn) and potassium silicate (25% Si and 10% K 2 O) respectively. Selected varieties of canola were sown in plastic cups containing 1500 g soil. Five seeds were sown in each cup and the air temperature at that time is, at day time 36.9 °C and night time 23.8 °C Latterly, only two healthy vigorous seedlings were allowed to grow, and rest were removed with thinning after 10 days of seed germination. The canola plant was grown up to 40 days and the all morphological, physiological and antioxidants were measured by using standard procedure.

Field Capacity
The canola seedlings were watered regularly after germination with 100% field capacity in order to give them salt stress. The salt stress (150 ppm) was applied to plant after the completion of germination. The salinity stress was applied after 10 days of germination and remain it for the period of three weeks. Seedlings were grown until vegetative stage for regarding different plant parameters.

Morphological Parameters
At the time of harvesting, the plants were removed from soil and placed in an empty container after thorough washing. After washing roots were placed on a filter paper to remove moisture. The root length was measured using a measuring scale. After that, one plant was picked from each replication for measuring root fresh weight using an electronic balance. Latterly, roots and shoot of canola plants were placed in oven at 70 °C for the period of two to three days until constant weight are obtained. The dry weight of roots was also recorded using an electronic balance. Similarly, the shoot length along with shoot fresh and dry weight was also recorded using same protocol as mentioned above.

Photosynthetic Pigments
In order to calculate the chlorophyll a, b, total chlorophyll and carotenoids contents, fresh canola leaves (0.5 g) were chopped into to 0.5 cm pieces, and then 5 ml of acetone (80%) was added. The absorbance of supernatant was measured at 645, 652 and 663 nm in spectrophotometer. Each of the chlorophyll contents were measured separately for the proper readings and observations by using the protocol given by [31]

Peroxidase Dismutase (POD)
The solution of POD in which the reaction occurs consists of 50 mM of phosphate buffer having a pH of 5, 20 Mm of guaiacol, 40 mM of H2O2, and 0.1 mL of enzyme extract. An increased absorption due to the formation of tetraguaicol at 470 nm was assessed after the 20 s interval. A single unit of enzyme is considered as the quantity of enzyme which mainly causes the raise in OD value with 0.01 in 1 min. This enzyme's activity was shown as units' min −1 g −1 [34].

Catalase (CAT)
The protocol devised by [34] was used for determination of catalase activity. Catalase was measured by measuring the alteration rate of water molecule and hydrogen peroxide from the oxygen molecule. The 3 ml sample solution comprises phosphate buffer at the rate of 50 mM. Their pH is neutral (7.0) not acidic and not a basic properties with 5.9 mM H 2 O 2 and an enzyme extract at the rate of 0.1 mL. The result was noted by the spectrophotometer by decline in absorption at 240 mm. these are consumption of H 2 O 2 after every minutes.

Ascorbate Peroxidase (APX)
To analysis the APX a method of monitoring the reduction in absorbance of ascorbic acid. The absorbance noted at 290 nm. The mixture of sample that containing 50 mM phosphate buffer with 7.6 ph. In 1 ml reaction and 0, 1 mN of Na EDTA, 0.25 mM of ascorbic acid, 12 mM H 2 SO 4 . This description method determine by the scientist [35].

Superoxide Dismutase (SOD)
The superoxide dismutase was measured according to the [36] method. Firstly took the plants and grinded with N 2 solution. If the plant grinded (convert into small pieces) then uniform by the phosphate buffer at the pH of 7.6. Then noted the result.

Statistical Analysis
Recorded data were arranged and subjected to analysis of variance techniques (ANOVA) using the Statistics 8.1 programming software. Thereafter, the least significant test at 5% probability was employed for determining the significance among treatment means [37].

Growth Attributes
The results revealed that salt stress significantly affected the measured all growth attributes such as root shoot length and their fresh as well as dry weight in canola presented in Table 1. The measured characters showed that the root length and their fresh as well as the dry weight of root of canola plant significantly enhanced where salt stress at the rate of 150 ppm was applied as compared to the control pot where no salt stress was applied in both varieties of canola.
Similarly the shoot length and their fresh as well as the dry weight of shoot of canola plant also reduced where 150 ppm salt stress was applied as compared to the control one in both varieties. The foliar application of silicon and zinc was significantly helpful for the improvement of root-shoot length and their fresh as well as the dry weight of both canola varieties under normal as well as the salt stress conditions. The highest performance was shown in the treatment where combination of foliar applied Si and Zn at the rate of 70 ppm in both conditions such as control without salt stress and salt stress at the rate of 150 ppm as compared to all others treatments while the minimum performance of treatment was observed in control treatment where no foliar was applied (Fig. 1a-f).

Photosynthetic Pigments
All photosynthetic pigments such as chlorophyll a, b, total chlorophyll contents and carotenoid contents of the both canola cultivars were substantially influenced under salt stress conditions (Table 1). Both varieties of canola are affected under the application of salt stress at the rate of 150 ppm as compared to the control where no salt stress was applied. The exogenous application of different treatments of silicon and zinc alone as well as combination significantly improved the all recorded photosynthetic pigments in both conditions such as normal where no salinity stress was imposed and under salinity stress at the rate of 150 ppm was imposed. The highest performance of treatment such as treatment No. T 6 where the combination of Si and Zn at the rate of 70 ppm was applied the three leaf stage as compared to all others treatment while the lowest values of photosynthetic pigments were noted in control treatment where no exogenous application of Si and Zn was applied under control as well as salt stress conditions (Fig. 2a-d).

Antioxidants
The results pertaining to antioxidants activity such as APX, CAT, POD and SOD differed significantly under the influence of foliar sprays, salinity and cultivars (Table 1). It was inferred that salt stress significantly affected the measured antioxidant contents for both verities of canola. Sandal cultivar remained superior as compared to variety 2 (Rachna).
The highest values of all measured antioxidants were noted under salt stress conditions as compared to the normal conditions where no salt stress was imposed. Among different treatments of exogenous application of Si + Zn at the rate 70 ppm significantly remained unmatched especially for sandal cultivar under normal as well as salinity stress conditions. Contrastingly, the lowest values of all recorded antioxidant assay was noted for control treatment where no foliar application of Si and Zn was applied (Fig. 3a, b, c & d).

Discussions
The results pertaining to the extrinsic impact of Si and Zn on canola cultivars under SS differed significantly. The SS had a drastic impact on all of the assessed parameters of canola,  . 1 (a-f). Effect of foliar applied Si and Zn on morphological attributes of maize under salt stress conditions. a Root length, b Root fresh weight, c Root dry weight. d Shoot length, e Shoot fresh weight f Shoot dry weight. (T 0 -control, T 1 -Si @ 35 ppm, T 2 -Si @ 70 ppm, T 3 -Zn @ 35 ppm, T 4 -Zn @ 70 ppm, T 5 -Si & Zn @ 35 ppm, T 6 -Si & Zn 70 ppm, V 1 = Sandal and V 2 = Rachna) Fig. 2 (a-d). including root-shoot length and fresh and dry weights along with leaf pigments composition as well as the antioxidants contents. However, co-application of Si and Zn (70 ppm dose) remained unmatched in terms of morphological traits of shoot and root which might be attributed to growth promoting effect of both elements [1,2,38]. Previously, it has been inferred that canola has evolved various systems to counteract NaCl toxic effects in order to withstand or evade adverse circumstances [30] which need to be ameliorated for achieving potential growth.
The results of the current study revealed that the shoot length and fresh and dry weight of canola were reduced under SS. These results was supported by [9] who opined that SS significantly reduced the shoot length and their fresh and dry weight. It was further inferred that plants causes closure of stomata which rendered plants unable to absorb the nutrients which restricted cell division. Due to this, the shoot length reduced due to unavailability of nutrient and water [15,39]. In a recent study, it was revealed Si and Zn application to canola increased shoot length and fresh and dry weight of salt-stressed plants. According to the findings of [25,40] who pointed out that foliar Si and Zn boosted shoot length in salt conditions by triggering cell division and maintaining optimum water status of the plants under saline conditions. These enter the leaf through the cuticle or certain apertures and aid in normal physiological processes and cell proliferation which increased shoot length [41,42]. Furthermore, [2,43] stated that stress diminished root length and fresh and dry weights of crop plants. It was attributed to stomata closure which made plant unable to acquire any nutrients in saline environment. When silicon and zinc are administered as foliar sprays, these penetrate effectively into plant tissues and triggered physiological processes which led to significant increment in the root length of salt stressed plants [25,44].
Chlorophyll pigments are the most vital components in leaf tissues for carrying out and maintaining the photosynthesis process. The results of the current study revealed that salinity stress decreased chlorophyll content compared to non-saline conditions. Our findings are comparable to those of [13], who found a corresponding decrease in chlorophyll pigments in various canola varieties subjected to SS. Foliar Si treatment decreased carotenoids degradation, and the leaf area of canola seedlings in duress rose considerably, indicating that it promoted sunlight radiation absorption which increased dry weight of plants. A reduction in chlorophyll concentration is caused by the formation of reactive oxygen species (ROS) in exposure of plants sunlight [45][46][47][48]. The production of ROS reactive oxygen species is caused by the mechanism of photosynthetic activity absorbing too much energy, which may be avoided by lowering chlorophyll levels. Our discoveries on chlorophyll content are consistent with several other research that have found a decrease in chlorophyll and carotenoids in crop plants exposed to salt toxicity [49].
The findings of this research, conveyed by [50], revealed that the vitality of leaves and roots in seedlings grown (control, salinity stress) significantly deteriorated. The quality of plant leaves in stressed plants were significantly damaged as a result of just this quiescent response. Since the photosynthetic rate was larger than the hydration, this passive outcome was seen in a seasoning blade. Extracellular treatment of Zn and/or Si at both levels (35 and 70 ppm) led to a tremendous change [40,51]. Independent variable Zn, Si, and related blends, on the other hand, can enhance the scenario of salinity-stressed canola fresh leaves and increase their pungency in terms of developing ROS. Under SS, the synergistic involvement of Zn and Si modulated SS might be attributed to better morphological growth and functioning of physiological process. Furthermore, Si can thicken the leaf and thus shield from maceration, and prevent water deficits [52,53]. Similarly the antioxidant activities such as APX, SOD, POD and CAT were significantly enhanced by Si exogenous application under saline conditions. The findings of this trial exhibited encouraging results regarding effectiveness of co-application of Si and Zn which resulted in significant improvement of antioxidant activities in canola cultivars under normal saline conditions. These findings are in concurrence with [10,16,54] who reported that the antioxidant activities in plant were enhanced by the application of Si under both normal and SS. These findings are also supported by the conclusions made by [25,42] who opined Zn and Si combine application could mitigate the adverse effects of salinity through morphological traits improvement and triggering the physiological processes such as biosynthesis of antioxidants which assisted crop plants to survive the adversaries offered by saline environment. However, these results offer an opportunity to serve as baseline to conduct further in-depth studies in order to evaluate other doses of Zn and Si under varying levels of salinity along with finding out the most optimum crop growth stage for foliar application of Si and Zn.

Conclusion
Salinity stress has seriously threatened the production of oilseed crops such as canola which has compromised the food and nutrition security. Fortification of foliar sprayed Si and Zn hold potential to mitigate the negative effects of saline environment by altering the morph-physiological mechanisms of canola plants. It has been inferred that canola cv. Sandal performed better under saline conditions, while co-application of Si and Zn (70 ppm) mitigated the deleterious effects of salinity by boosting plants morphological traits and physiological functioning of canola plants. These encouraging results necessitate conducting further in-depth studies involving different doses and sources of Zn and Si for ameliorating the adverse effects of salinity on canola growth and productivity.