Foliar application of potassium on antioxidant enzyme activities of tomato plants under drought stress

Water stress negatively affects productivity in crops, while the foliar application of potassium­containing compounds may be helpful in reducing the drought effects. This study evaluated the efficacy of foliar applied potassium chloride (control ­ distilled water spray ­, 3 and 6 mM­1) on tomato plants under drought stress. Three irrigation levels were maintained at 100, 75 and 50% according to evapotranspiration designated as well watered, moderate and severe drought stressed. Increasing drought stress significantly reduced plant growth and yield. The foliar applied KCl produced maximum leaf area, stem diameter and length, plant yield under each drought stress conditions compa­ red to control. The minimum of growth factors were obtained by control under severe stress. Highest yield per plant was also recorded for foliar applied KCl under moderate condition than other treatments. Foliar applied KCl alone decreased the SOD, CAT and PPO in well­watered condition but KCl application on tomato plants under drought stress induced the antioxidant enzyme activi­ ties more than control well­watered treatment.


Introduction
The major limitation for plant growth and crop production in arid and semiarid regions is soil water availability. Plants that are continuously exposed to drought stress can form ROS (Reactive oxygen species), which leads to leaf damage (Foyer et al., 2002;Oerke and Dehne, 2004;Cakmak, 2005) and, ultimately, decreases crop yield. The decrease in soil water potential causes alteration in minerals uptake by plant roots and reduc tion in leaf expansion under drought stress conditions (Kaminek et al., 1997;Pospisilova et al., 2000).
Drought is becoming a serious threat to crop production worldwide resulting in 67 and 82% reduction in K uptake under mild and severe water stress conditions (Baque et al., 2006). During drought stress, root growth and the rates of K + diffusion in the soil towards the roots were both restricted, thus limiting K acquisition. The resulting lower K concentrations can further depress the plant resistance to drought stress, as well as K absorption, which ultimately leads to reduction in the fruit size, plant yield, lack in red color of tomato and fruit quality (Bidari and Hebsur, 2011;Afzal et al., 2015), and deteriorated photo synthesis, enzyme activation in plants (Marschner, 1995;Garg et al., 2004;Afzal et al., 2015). Maintaining adequate plant K is, therefore, critical for plant drought resistance.
When plants were supplied with different K + con centrations and then subjected to drought stress, their stomatal conductance was more markedly reduced in normal K plants than in low K plants (Wang et al., 2013). During drought stress, the sto mata cannot function properly in K + deficient plants, resulting in greater water loss. Drought stress did not decrease water use efficiency (WUE), whereas it did increase WUE by rapid stomata clo sing during water deficit (Egilla et al., 2005). Adequate levels of K nutrition enhanced plant drou ght resistance, water relations, WUE and plant growth under drought conditions (Wang et al., 2013). Besides various adaptive mechanisms; potas sium (K) sprayed under drought condition can improve the tolerance of crop plants to various types of abiotic stresses, and it also improved sub sequent growth and yield. Mengel and Kirkby (2001) reported that K improves physiological processes by the regulation of turgor pressure and photosynthe sis; translocation of cations and enzymes activation, while, Cakmak (2005) also observed that plant suf fering from drought stress required more internal K. Yield limiting effect of water deficit could be over come by increasing K supply (Damon and Rengel, 2007). In legumes, devastating effects of drought can be alleviated by rich K supply (Sangakkara et al., 2000). A close relationship between K nutritional status and plant drought resistance has been demonstrated.
The bottom lines of the reviewed results in this section indicate that under drought stress conditions, yield losses can be minimized by the sufficient supply of K. However, its application effect at tomato growth stages is not well understood yet. The objec tive of present work was to study the possible role of K applied on tomato plant under drought, in mitiga tion of stress in terms of physiological components and antioxidant enzyme values.

Materials and Methods
This experiment was designed to observe the effect of KCl on tomato (Solanum lycopersicum L.) seedlings under drought stress. Seeds of tomato were sown in plastic trays and maintained in a greenhouse up to 4 real leafy stages, at Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran. The experimental design was a split plot design with three water stress plot as main plot and 3 KCl treatments (0, 3 and 6 mM 1 ) as subsidiary plot with 3 replications. The subsidiary plot area was 1 m 2 (1×1 m) and consisted of four plants per plot. After 70 days from sowing, seedlings with uniform growth were transplanted to an experi mental field with a 50 cm interseedling spacing. According to evapotranspiration (ET c = crop evapo transipration), the water stress was conducted on tomato plants at three levels wellwatered (100% ET c ), moderate and severe drought stress (75 and 50% ET c , respectively). According to Penman Monteith equation, the crop evapotranspiration, ETc (Formula 1), is calculated by multiplying the referen ce crop evapotranspiration, ETo, by a crop coeffi cient, Kc and ETo (Formula 2)  Where Kp= Pan coefficient (0.77), Ep0 pan evapora tion. The foliar spray was applied five times (during two month) to tomato plants during growth and fruit set with KCl at 0, 3 and 6 mM 1 dose.

Growth and plant analysis
Plant height and stem diameter were measured at the end of the harvesting season and presented as cm and mm respectively. The total yield of tomato fruits were measured by gram scales (g) in different harvesting times per plant. The leaf area (LA) measu red by Windias (DeltaT Co, England) and presented as (mm 2 ).
The integrated wateruse efficiency (formula 3) is typically defined as the ratio of biomass produced (D, kg h 1 ) to the rate of total water irrigation (W i , m 3 ha 1 ) and rainfall (W p , mm) during the drought stress treat ment.

Assays of enzymatic and non-enzymatic antioxidants
Fruits were randomly selected from each treat ment, at the end of the experiment. Total soluble proteins were quantified by following the protocol devised by Bradford (1976). For determination of enzymatic antioxidants, fruit samples were extracted in 50 mM phosphate buffer (pH 7.8). The extract was centrifuged at 15,000 rpm 4°C and the supernatant was used for further assay of catalase (CAT) Chance and Maehly, 1955 and poly phenol oxidase (PPO) (Siriphanich and Kader, 1985) and super oxide dismu tase (SOD) activities (Giannopolitis and Ries, 1977). Tomato juice was squeezed from the fresh tomatoes onto a digital refractometer (PR100, Atago Co. Ltd., Tokyo, Japan) to measure total soluble solids (TSS) and the results were expressed in Brix according to AOAC method 932.12 (2005). For measured the proli ne content, leaves were randomly selected and expe rimented according to Bates et al. (1973) method.

Statistical analysis
Effects of water stress treatments; KCl and corre sponding interactions were determined by analysis of variance according to the general linear model proce dure of SAS (version 8.2; SAS Institute, Cary, N.C.). Means were compared using Least Significant Difference (LSD, p≤0.05) according to method descri bed by Tukey HSD. Analysis showed a significant inte raction between KCl and watering treatment for the some measured parameters. The graphs draw by using excels 2010 software.

Results
Increasing drought stress (DS) levels significantly reduced plant growth and yield, but foliar applica tions of KCl improved the harmful effects of drought stress.
Comparison of means indicate that leaf area (LA) was gradually decreased with increasing drought stress (Table 1). The minimum leaf area was obtained under extreme drought conditions (50% ETc). Water stress decreased LA but exogenous application of KCl ameliorated the negative effects of water stress significantly than water spray only (Fig. 1a). However, highest LA in each treatment was recorded for foliar applied KCl than control under wellwatered, mode rate and severe drought stress. A minimum LA was recorded under severe drought stress, especially con trol × 50% ETc treatment (Fig. 1a).
The increasing water stress decreased stem length (Fig. 1b). A maximum stem length was observed under wellwatered conditions by exogenous applica tion of 6 mM 1 KCl while in moderate and severe drought, foliar applied KCl had no significant effects. Minimum stem length was noted under extreme water stress without foliar KCl application. Stem dia meter decrease in response to DS (Table 1). Foliar KCl application helps to plant tolerance with increasing stem diameter under DS condition. Data showed (Fig.  1c) the increasing in KCl doses (0 up to 6 mM 1 ) lead to increasing in stem diameter in all of treatments.
The tomato yield displayed a significant reduction in response to the increasing levels of water deficit treatments. For example, under effect of 50% ETc condition, the yield decreased by 21% (Table 1) com pared to control. In the other hand, plants had more vegetative growth and less yield in wellwatered con dition (100% ETc) than moderate DS (75% ETc). In nonDS condition, foliar spray of KCl showed similar fruit yield under wellwatered, moderate and severe drought conditions. But foliar KCl application had effective enhancement on plant yield under drought stress (Fig. 1d). Tomatoes irrigated with 50% ETc without KCl foliar application also produced fruits with significantly higher juice brix value than other  (Fig. 1e). The results in figure 1 (f) revealed that DS on tomato had higher significantly record in water use efficiency than those normally irrigated. Because of fewer yield in control treatment than 75% ETc, the results affirmed that the treatment 75% ETc and then control showed the highest water use efficiency than 50% ETc treatments. Foliar application of KCl had no significant effects of WUE under nonDS condition. Finally, the foliar application of KCl × 75% ETc showed the highest significant record in WUE in plants subjected to DS, respectively. Meanwhile, the treat ment 6 mM 1 KCl × 75% ETc recorded the highest significant WUE compared to other studied treat ments under wellwatered and sever DS.
Leaf proline concentration responded differently to drought and K supply. Drought stress (Table 1) increased proline concentration in leaves but exoge nous KCl decreased the proline content under none DS conditions ( Table 2). The tomato leaves were sprayed with KCl had more proline content under drought stress than control conditions (Fig. 2 a). At 0 mM 1 KCl level with no DS, the proline content in lea ves was 1168.4 µM/g F.wt. which increased to 13368.5 and 12111.5 µM/g F.wt. with increasing DS stress in 50% ETc with 3 and 6 mM 1 KCl foliar applica tion, respectively.
Increase in KCl doses enhanced the total soluble proteins in tomato fruits (Table 2). Maximum fruit protein content was evident at moderate stress with 3 mM 1 KCl followed by 0 mM 1 KCl with minimum value under wellwatered conditions (Fig. 2b). Foliar spray with KCl had impressive effects on protein con tents under wellwatered and drought stress. However, the exogenous application of KCl perfor med better than control for fruit total soluble pro tein.
A rise in drought stress also amplified the antioxi dants status of tomato fruits (Fig. 2c, d, e). Results showed that the enzymatic antioxidant contents in tomato fruits were sprayed by KCl, were decreased. So, the maximum SOD with no significant differences were recorded for foliar applied 0 and 3 mM 1 KCl under severe drought and 0 mM 1 KCl under modera te conditions (Fig. 2c). Increased CAT activities with no significant differences were found under medium and severe drought stress (Fig. 2d). Decreasing water level increased PPO contents for all treatments (Fig.  2e) and the highest value of PPO was obtained under sever DS but increasing in foliar application of KCl doses from 0 to 6 (mµ 1 ) caused to decrease the PPO fruit content (Fig. 2e).

Discussion and Conclusions
Increasing water stress has direct impact on crop growth and yield reduction and similar observations for reduction in growth and fruit yield of tomato were found in present study. Reduction in tomato yield under restricted water availability might have been due to reduction in photosynthetic area such as leaf area and leaf number (Chaves et al., 2011;Khan et al., 2015). In the present study, yield, leaf area and stem length were drastically reduced due to drought effect, whilst KCl foliar application improved these characters in tomato plants.
It has been reported that the foliar application of KCl improved the growth factors in agronomy crops (Ahmad and Jabeen, 2005;Yasmeen et al., 2013). Besides adaptation role, positive effect of KCl on pro tein contents was also pronounced under drought stress. Proline is a wellknown amino acid that gene rally accumulates when plants are exposed to envi ronmental stresses (KaviKishor et al., 2005). Enhanced proline synthesis is a common response of tomato plants to drought and may determine the stress tolerance (Doan and Maurel, 2005;Khan et al., 2015). Proline is believed to acts as a signaling mole cule that initiates adaptation to the stress (Maggio et al., 2002), acts as osmolyte for osmotic adjustment (Hayat et al., 2012), helps in stabilizing membranes/proteins and scavenges free radicals (Ashraf and Foolad, 2007). Thus, it decreases the adverse effects of cytoplasmic acidosis and maintains proper NADP + /NADPH ratios (Liang et al., 2013). In plants grown under drought conditions, proline indu ces the expression of drought stress responsive genes and, thus, decreases the damage due to exces sive Na + ions accumulation (Chinnusamy et al., 2005). Proline act as a compatible solute in the plants (Mansour, 2000) and, generally, increases with increase in both the salinity stress and drought stress duration (Kishor and Sreenivasulu, 2014). Thus, it is likely to observe enhanced proline synthesis with increasing drought stress duration. Antioxidant enzy mes activities were considered as indicators of sca venging ROS and reducing oxidative stress (Dionisio Sese and Tobita, 1998;Lin and Kao, 2002). For exam ple, SOD may convert superoxide radicals into H 2 O 2 and H 2 O 2 was further decomposed by CAT and POD (Redman et al., 2011). In this study, drought stress significantly increased the antioxidant enzymes acti vities. The results of many researchers' studies pro ved that adequate external K supply significantly decreased antioxidant enzymes activities and proline in drought stressed plants might be caused by enhan cing plant physiological metabolism and reducing ROS production and MDA content (Wei et al., 2013;Yasmeen et al., 2013;BahramiRad and Hajiboland, 2017). In the other hand, the combination of drought stress and exogenous KCl application improved the antioxidant activities and proline content than well watered conditions. So, rise in drought stress with exogenous application of KCl induce tolerance in crops (Yasmeen et al., 2013). A similar trend was fol lowed by all the treatments in case of antioxidant enzyme activities under each water stress treatment.
The results of this study clearly demonstrated that water deficit at any critical crop growth stage seve rely affected the physiological and antioxidant and nonantioxidant parameters of tomato. Foliar appli cation of K on a drought stressed plants at all growth stages improved the physiological performance and plant tolerance but reduced antioxidant enzyme acti vities. All these findings lead us to recommend that for tomato crop under drought farmers should spray the crop with 6 (mµ 1 ) KCl to minimize the negative effect of drought. This can have a dual benefit: improving the physiological performance of tomato and supplying K nutrient to plants. For the foliar spray on a small scale, a common handboom sprayer can easily be used, whereas on large scale use of a mechanical boomsprayer is advised.