Defensive Impact of Foliar Applied Potassium Nitrate on Growth Linked with Improved Physiological and Antioxidative Activities in Sunﬂower ( Helianthus annuus L.) Hybrids Grown under Salinity Stress

: Salt stress is recognized to negatively inﬂuence the fundamental processes in plants regarding growth and yield. The sunﬂower ( Helianthus annuus L.) is considered an important industrial crop because of the good quality of oil it produces that can be used for cooking purposes. The exogenous application of potassium (K) has been reported to enhance abiotic resistance and increase yield in crops. Here, we explored the impact of foliar-applied K at 500 ppm on the physiological and biochemical traits, antioxidant activities, and growth attributes of sunﬂower grown under salt stress (140 mM NaCl). The ﬁndings indicated that salinity stress adversely affected photosynthesis and various gas exchange characteristics. Foliar applied K markedly improved the stomatal conductance, transpiration rate, water use efﬁciency, CO 2 assimilation rate, total soluble proteins, chlorophyll pigments, and upregulated antioxidant system, which are responsible for the healthy growth of sunﬂower hybrids grown under salinity stress. The shoot and root lengths, plant fresh and dry weights, and achene weight were signiﬁcantly increased by K application. Overall, foliar applied K signiﬁcantly improved all of the aforementioned attributes and can attenuate the deleterious inﬂuences of salinity stress in sunﬂower.


Introduction
The intensification of crop production in semi-arid regions of the world requires vigorous and healthy crop growth and establishment in order to overcome the adverse effects

Experimental Details
A pot experiment was conducted to cope with the salinity stress in sunflower through the foliar application of K under greenhouse conditions at the Old Botanical Garden, University of Agriculture, Faisalabad, Pakistan. This experiment was conducted in a completely randomized design (CRD) with a factorial arrangement having three replicates. The seeds of sunflower hybrid L-16003 and L-Check 1 were purchased from Punjab Seed Corporation, Faisalabad, Pakistan. Plastic pots, 30 cm in height and 22 cm in diameter, were filled with growth media, with 5 kg in each pot. The growth media consisted of compost, clay, silt, and sand in equal proportions. Four seeds were sown in each pot and the pots were placed in a greenhouse. After 15 days of emergence, thinning was done to maintain a single plant in each pot for further experimentation. Pots were divided into main groups according to fresh irrigation water (4 mM NaCl) and saline solution (140 mM NaCl). Pots were irrigated at field capacity (FC), and FC was maintained up until harvesting of the plants, starting from the sowing. In order to determine the water loss by transpiration, the pots were weighed on daily basis and the soil moisture contents were sustained by recompensing this water loss through the addition of tap water up to the initial weight of the respective pot. The gravimetric method, described by Nachabe [34], was followed to determine the FC. Only once throughout the course of experimentation, salinity treatment of 140 mM NaCl was applied (at three leaf stage) to compare the fresh water irrigation and be used as a control. Initially, 150 mL of water was required for FC as each pot contained 5 kg of growth media. Then, 8.775 g of NaCl was dissolved in 150 mL of water to prepare a 140 mM solution of NaCl, as the NaCl molar mass is 58.5. After that, the pots were irrigated with fresh water. The first round of foliar spray of KNO 3 at a 500 ppm concentration was applied after one week of imposition of salinity treatment. During experimentation, KNO 3 was applied three times with an interval of one week after the application of the first round. According to the water requirements of the crop, the pots were irrigation throughout the course of experimentation. Water spray was also considered as a control treatment.

Biochemical, Mineral, and Antioxidant Enzyme Activity Measurement
The data regarding biochemical and antioxidant activities were recorded after 5 days of the third round of treatment application. Chlorophyll a (663 nm) and b (645 nm) contents and carotenoids (470 nm) were measured through the extraction of leaves in an 80% solution of acetone, according to Arnon [35], by using Spectrophotometer with the following formula where V is the volume of the extract (mL) and W is the weight of the fresh leaf (g).
Soluble phenolics were determined by Julkunen-Titto's method [36]. A 0.1 g plant sample was extracted in 1 mL acetone (80%). After centrifugation for 15 min at 12,000 rpm, the supernatant was collected in microfuge tubes and stored at −30 • C until it was used. In a test tube, 100 µL supernatant (diluted it up to 1 mL), 0.5 mL of folin phenol reagent (Fluka), and 2.5 mL of 20% sodium carbonate (Na 2 CO 3 ) were added and made the volume up to 5 mL. Then, the reaction mixture was vortexed for 5-10 s and kept for 20 min at room temperature. The absorbance of the colored complex was noted at 750 nm, and 80% acetone was used as a blank.
The Na + , K + , and Ca 2+ concentrations were meacured using a flame photometer (Sherwood, UK, Model 360) according to the standard procedure of the USDA Laboratory Staff [37]. The activities of the peroxidase (POD) and catalase (CAT) readings were noted on a spectrophotometer by the procedure of Chance and Maehly [38]. The Bradford technique [39] was used to determine the total soluble protein. For the measurement of the gas exchange features, the flag leaf was used during 10:00 a.m. to 12:00 p.m. Stomatal conductance (gs) (µmol m −2 s −1 ) and transpiration rate (E) (µmol m −2 s −1 ), as well as the CO 2 assimilation rate (A) (µmol m −2 s −1 ), were estimated according to the procedure described by Long [40], using an infrared gas analyzer (IRGA) LI-6400 portable device. Water use efficeiency (WUE) was calculated by dividing the A with E (A/E). The rate of water loss and rate of CO 2 uptake were used to assess the rate of transpiration and photosynthetic carbon assimilation, respectively, using a working principle of LI-6400.

Growth Attributes
The number of leaves plant −1 were calculated manually. The formula to calculate the leaf area (cm 2 ) was as follows: Leaf area = leaf width × leaf length × correction factor (0.75) The leaf width and length were measured manually. The leaf length was considered from the leaf base to the tip of the leaf and the width was the maximum broad portion. The shoot (cm) and root (cm) lengths of the sunflower plants were measured with the help of a meter rod, and the mean values were calculated. The shoot was considered from the base to the top of the plant. Similarly, the root was considered from base (underground portion) to the tip of the root/root hairs. The achene weight was measured by electric weigh balance. The fresh weights of the roots (g) and shoots (g) were measured with an electric weigh balance and the sum for the plant fresh weight and dry weight were calculated. The roots and shoots were dried in sunlight for 3 days after this and were kept in the oven at a temperature of 65 • C until reaching a constant weight.

Statistical Analysis
For each parameter, ANOVA was applied to the data, which were organized in a completely randomized design (CRD) [41]. Co-Stat V6.3 computer software (Cohort software, Berkeley, CA, USA) was used for the data analysis. Microsoft Excel was used for the graphical presentation.

Growth Attributes
The growth parameters of the sunflower hybrids were significantly affected by the foliar application of K grown under normal and saline conditions (Table 1). Sunflower L-16003 hybrid produced a maximum number of leaves plant −1 under normal conditions where K was applied ( Figure 1a). The foliar application of K was also effective under normal and saline environments. The L-check1 hybrid produced the maximum leaf area under normal and saline environments through the foliar supplication of K as compared with the L-16003 hybrid (Figure 1b). A similar trend was also observed regarding shoot length (Figure 1c). Foliar application K was more effective regarding root length. The maximum root length was recorded in the L-16003 hybrid even under a saline environment (Figure 1d), while the minimum root length was noted in the L-check1 hybrid under a saline environment. More plant fresh and dry weights were recorded under a normal environment with the foliar application of K in the L-16003 hybrid (Figure 1e,f). L-check1 hybrid performed better regarding achene weight both under normal or saline environments. The foliar application of K was also effective regarding achene weight in both environments ( Figure 1g). The minimum achene weight was found in the L-check1 hybrid under a saline environment without exogenous treatment. SOV-source of variance; DF-degree of freedom; WUE-water use efficiency; AR-assimilation rate; TSP-total soluble proteins; NS -non-significant; *-significant at p ≤ 0.05; **-significant at p ≤ 0.01.

Biochemical and Physiological Attributes
Biochemical and physiological attributes including stomatal conductance, transpiration rate, water use efficiency, CO 2 assimilation rate, total soluble proteins, and chlorophyll a and b contents were significantly affected under a saline environment compared with normal conditions (Tables 1 and 2). The data regarding the stomatal conductance of two sunflower hybrids grown under control and saline environments is presented in Figure 1h. Saline conditions significantly reduced the stomatal conductance. In the L-16003 hybrid, the maximum reduction in gas exchange attributes was noted under the saline condition when K was not applied. Conversely, significant results were observed in the L-chek1 hybrid under the non-saline condition with K application. Salt stress significantly reduced the transpiration rate ( Figure 2a). In the L-16003 hybrid, an extreme reduction was observed under the saline condition when K was not applied. Nevertheless, a less non-significant content was observed under the control conditions with K application in Line-chek1.
On the other hand, the spray of K showed non-significant results, but the difference between hybrids was significant. Salt stress reduced the water use efficiency (Figure 2b). Under salinity conditions when K was not applied, the maximum reduction was noted in Line-16003. Conversely, in the L-chek1 hybrid, the maximum non-significant increase was perceived under the controlconditions. Overall, L-chek1 hybrid performed better than the L-16003 hybrid. A maximum decrease was noted in Line-chek1 under salinity stress when K was not sprayed. Generally, the spray of K showed non-significant results, but the hybrid difference was significant. The data related to the total soluble protein of two sunflower hybrids tested under the control and salt stress conditions are given in Figure 2d. Salt stress significantly reduced the total soluble protein. In the L-16003 hybrid, the maximum reduction was noted under saline conditions when K was not applied. Overall, the L-chek1 hybrid performed better than the L-16003 hybrid.  Transpiration rate (µ mol m −2 s −1 ) Water use efficiency (µ molCO2/mmol H2O) CO2 Assimilation rate (µ mol m −2 s −1 ) Total soluble proteins (unit/mg fresh weight) Chlorophyll a contents (mg/g fresh weight) Chlorophyll b contents (mg/g fresh weight) Phenolics (mg/g fresh weight) Carotenoids (mg/g fresh weight) Data about the chlorophyll a and b contents of the two sunflower hybrids tested under non-saline and salinity stress are presented in Figure 2e,f. In chlorophyll a, a significant decrease was reported under saline stress conditions. Under saline stress conditions, when K was not applied, a maximum reduction was found in the L-16003 hybrid. Conversely, the most significant increase was noted in both hybrids of sunflower under non-saline conditions when K was applied in the L-chek1 hybrid. The spray of K showed a significant result, but the hybrid difference was observed as being non-significant. Regarding the chlorophyll b contents, a similar trend was observed. Overall, the L-16003 hybrid performed better than the L-chek1 hybrid.

Antioxidant and Enzymatic Activities
The total phenolics and carotenoid concentrations were similar within the hybrids, while they were significantly affected by K application and a saline environment ( Table 2). Saline stress indicated a significant rise in phenolic (Figure 2g). The minimum content of phenolic was observed in the L-16003 hybrid under the normal conditions when K was not applied. Under salinity conditions, a maximum increase was observed in the L-chek1 hybrid. Effect of the foliar application of K was significant in both hybrids. The combination of salt with K also showed significant results. For instance, the L-chek1 hybrid performed well as compared with the L-16003 hybrid (Figure 2g). Data about the carotenoids of two sunflower hybrids were tested under the control, and the salinity stress situation is reported in Figure 2h. Salinity stress significantly reduced carotenoids. Under saline conditions, more reductions were found in the L-chek1 hybrid. Conversely, a maximum significant increase was seen in the L-16003 hybrid under non-saline conditions when K was applied. Overall, the L-16003 hybrid performed better than the L-chek1 hybrid.
Catalase activities were significantly affected by the foliar application of K under normal and saline environments, even within the hybrids ( Table 2). In contrast, the peroxidase activities were only affected by the saline environment. Under saline stress conditions, when K was not applied, a maximum rise was seen in the L-16003 hybrid. Under control conditions, a maximum reduction was recorded in the L-chek1 hybrid (Figure 3a). Significant results were observed in the hybrids as a result of foliar applied K. Data regarding the peroxidase of both sunflower hybrids tested under control and salt stress conditions with foliar applied K are presented in Figure 3b. The application of salt stress indicated a significant increase in peroxidase activity. In the L-chek1 hybrid, a maximum increase was noted under saline stress conditions when K was not applied. A maximum reduction was recorded in the L-16003 hybrid under non-saline conditions. Overall, the L-chek1 hybrid achieved better results than the L-16003 hybrid.

Mineral Homeostasis
The root calcium was not significantly affected by the saline environment and even within the hybrids, but foliar application K significantly affected the concentration of calcium in the roots (Table 2) while the shoot calcium was significantly affected by K treatment and salinity stress. The root and shoot K was significantly affected by foliar treatments and salinity stress ( Table 2). The root sodium was not significantly affected by the foliar treatment, but were significantly affected within the hybrid and by foliar treatment (Table 2). In contrast, shoot sodium was significantly influenced by salinity stress and foliar treatments even within hybrids ( Table 2). Under salinity stress, a non-significant decrease in root Ca 2+ was observed (Figure 3c). Under saline conditions, the maximum reduction was examined in L-16003 when K was not applied. Nevertheless, a significant increase was detected in the sunflower L-chek1 under control conditions when K was applied. Salt stress significantly reduced the root Ca 2+ as shown in Figure 3d. In the L-16003 hybrid, the maximum reduction was perceived under the saline stage. A maximum significant increase was examined in the L-chek1 hybrid under the control conditions when the K was applied. Alternatively, the foliar application of K showed significant results, but non-significant results were recorded for the hybrids.

Catalase (unit/mg protein)
Peroxidase (unit/mg protein) Root Ca 2+ (mg/g dry weight) Shoot Ca 2+ (mg/g dry weight) Root K + (mg/g dry weight) Shoot K + (mg/g dry weight) Root Na + (mg/g dry weight) Shoot Na + (mg/g dry weight) Data relating to root the K + of two sunflower hybrids is indicated in Figure 3e. Salt stress indicated a non-significant reduction in root K + . In the sunflower L-16003 hybrid, a maximum reduction was detected under salt stress when the applied concentration of K was nothing. Overall, the L-chek1 hybrid performed better than the L-16003 hybrid. The shoot K + was decreased significantly by the salt stress (Figure 3f). An extreme reduction was examined in L-16003 under saline conditions without K application. Although a maximum significant rise was observed in the L-chek1 under the control condition when K was applied as a foliar spray. In other words, the spray of K shows significant results, but non-significant results were recorded regarding the hybrids. The presence of salt stress indicated a significant reduction in root Na + (Figure 3g). Under saline conditions when K was not applied, a maximum reduction was noted in the sunflower L-chek1 hybrid. Nevertheless, a maximum non-significant increase was observed in L-16003 under nonsaline conditions when K was applied. Alternatively, the spray of K showed non-significant results but a significant effect regarding hybrids. Overall, L-16003 performed better than the L-chek1 hybrid. Salt stress application triggered a non-significant reduction in the shoot Na + . Under saline conditions, when K was not applied, a maximum reduction was noted in the L-16003 hybrid. However, a maximum increase was perceived in the L-chek1 under control condition when K was folairly applied. On the other hand, the spray of K indicated significant results. K hindered the salt effect. Overall, the L-chek1 hybrid performed better comparatively than the L-16003 hybrid (Figure 3h).

Discussion
Salinity is a key stress limiting agricultural productivity worldwide. Salinity stress induced the nutritional imbalance and decreased the dry weight production in both hybrids as the present study was planned with aims to improve sunflower growth attributes under salinity stress by exogenously applied macronutrients like K. Salt stress-induced a nutritional imbalance [5] and a reduction of growth and productivity [8] have been observed in amaranth, which corroborated the present findings. The findings of our study were supported by Hussain et al. [42] and Shehzad et al. [18]. The findings of the current study regarding the leaf area, shoot length, and achene weight were in harmony with the outcomes of different crops observed by Latef and Chaoxing [43] on pepper and Mostofa et al. [44] on rice. The reduction effect of salinity stress on plant growth parameters may be due to the toxic effect of Na and Cl ions, and their excessive accumulation around the root system and accordingly in plant cells. Kosová et al. [45] explained that these ions reduced the uptake of water by plants resulted from the increased osmotic pressure of soil solution. Rehman et al. [26] and Farooq et al. [46] also repoted that the application of minerals either alone or in combination with growth promoters improved the growth attributes of crops.
Plants overcome the salinity toxic effect via mechanisms of osmotic adjustment (increases the compatible solutes), improved stomata closure, and decreased photosynthesis rates. Foliar applied K enhanced the growth parameters by improving the osmotic adjustment [47]. K sustains the turgidity of the guard cells, opening and closing of the stomata, and maintains water use efficiency [48]. When salt was applied on sunflower replicates, the negative effects were observed as a decrease in leaf area (cm 2 ), but when K was foliar applied, it suppressed the effect of NaCl as K inhibited the salt effect. According to Ashfaq et al. [49], K is an essential macronutrient that improves stress resistance in plants and also plays role in osmotic adjustment. The foliar application of K by 500 ppm increased various growth attributes like the number of leaves, plant length, leaf area, fresh and dry plant weight, and capitulum size compared with the control. Zaman et al. [16] reported that K + is parallel to Na + , many K transporters do not distinguish between these cations. Salinity indicated an adverse accumulation of sodium ions in the plant cell. Therefore, the application of K at a higher concentration is beneficial for the growth of sunflower. Furthermore, the foliar spray of K also positively adjusts the membrane stability and turgidity of plants under salt stress, that is why plants should be treated with K in both conditions either in the absence and presence of stress conditions. Raza et al. [15] reported that the foliar application of 1% K enhanced all of the parameters relevant to the morphological, physiological, and biochemical processes in sunflower.
Salt stress induces an over production of ROS that elicits DNA damage, inhibition of photosynthesis, lipid peroxidation, reduction in the rate of photosynthesis, and disturbance in the mineral nutrient status [50]. Sodium is the major cation (Na + ) that accumulates mainly in the roots and stems as the salt concentration increases in the rhizosphere [51]. Tabaxi et al. [52] stated that the application of various fertilizers or mineral elements improved the agronomic and quality charateristics of crops. Jan et al. [14] concluded that the valuable macronutrient K triggers the growth of sunflower, and its yield becomes good because it decreases the transpiration rate and enhances the water uptake. It also prolongs the turgidity and minimizes the adverse effect of reactive oxygen species. In sunflower, K plays a very important role in salinity tolerance. It increases growth and minimizes the effect of salinity [53]. K is a good osmoticum and charge carrier in the plant and antagonistic relationship with a sodium uptake. In a saline environment, K + can be used as a metabolite to control the turgor and avoid osmotic shock [54]. It is an essential macro element of plant growth and plays a vital role in several metabolic activities, i.e., the flow of water, nutrients, and carbohydrates in plant tissue. K can counteract the harmful effect of salinity and maintains the ions' homeostasis. The application of inorganic fertilizers and mineral elements is considered a helpful practice in maintaining the crop productivity with improved soil fertility to achieved the maximum plant growth and economical yield under stressful conditions [55,56].
In the present study, salinity stress enhances the total phenolic contents of sunflower in both hybrids. Interestingly, potassium application also accelerates the augmentation of the total phenolic contents. The literature has shown that plants have both antioxidant enzymes and secondary metabolites and/or non-enzymatic antioxidants, such as phenolics, for example hydroxybenzoic acids [57], hydroxycinnamic acids [58], flavonols [59], flavanols [60], flavanones [61], flavones [62], betalain [63], tocopherols [64], betacyanin [65], ascorbic acids [66], chlorophyll a [67], carotenoids [68], chlorophyll b [69], betaxanthin [70], and beta-carotene [71], with a strong radical scavenging capacity [72]. Nevertheless, for stress homeostasis, abiotic stress-induced plants have evolved mechanisms to enhance the concentration of the majority of secondary metabolites and/or antioxidants [58,73] and detoxify the ROS. Akram et al. [74] applied 150 mM sodium chloride on sunflower, and it influenced the osmotic potential in leaves but significantly enhanced the catalase, superoxide dismutase (SOD), and peroxidase activity. Similarly, salinity stress promotes reactive oxygen species (ROS) accumulation to adversely affect the plant developmental processes [75]. The shoot and root Ca 2+ were decreased through the application of salinity stress, and a maximum reduction was examined in L-16003. Salinity increased the uptake of Na+ and reduced K+ and Ca2+, but the foliar application of K enhanced the concentration of K+ and Ca2+ in the root and shoot while it reduced the uptake of Na under saline conditions (Figure 3c-h). Our findings are in line with Hurtado et al. [12], who stated that salinity adversely affects the uptake of nutrients in sunflower. The decreased uptake efficiency of nutrients in sunflower plants under salinity stress may have been caused by Na + competing with cations, a process that is associated with toxic effects such as the generation of reactive oxygen species, which can degrade enzymes or nutrient uptake transporters [76].

Conclusions
The growth, physiological, gas exchange attributes, antioxidants, and Ca 2+ and K + balance were significantly enhanced by the foliar application of K at 500 ppm under a saline environment. A healthy growth of sunflower was also achieved through foliar applied K. Further studies are required to explore the mechanism of K at a molecular level in sunflower as well as in other field crops.

Data Availability Statement:
The data that support the outcomes of the current experimentation are available from the corresponding author upon reasonable request.