Abscisic Acid (ABA) mitigates drought stress in sunflower by enhancing water relations and osmotic adjustments

Drought stress significantly alters plant growth by disturbing plant water relations. Production and accumulation of compatible solutes improve plant water relations and help the plants to survive under water-limited conditions. Abscisic acid (ABA) could ameliorate the negative consequences of drought stress. Therefore, this two-year field study was conducted to infer the role ABA application (0, 5 and 10 μM) at bud initiation (BBCH-51) and flower initiation (BBCH-55) in improving plant growth under water deficiency imposed at various growth stages. Drought stress was imposed at BBCH-51 and BBCH-55 by skipping irrigation, whereas a well-watered treatment was also included for comparison. The water relations, accumulation of compatible solutes and achene yield were significantly reduced by drought stress imposed at BBCH-51 and BBCH-55. The negative impacts of drought stress were more prominent at BBCH-55 than BBCH-51. Hence, BBCH-55 proved more sensitive growth stage to drought stress than BBCH-51. The ABA application under stress-free conditions significantly reduced achene yield, with more reduction with higher ABA level. The ABA application of 5 μM either at BBCH-51 or BBCH-55 improved plant water relations, accumulation of compatible solutes and achene yield under drought stress whereas 10 μM ABA application was less effective. Therefore, it is recommended that 5 μM ABA application could ameliorate the negative impact of water deficit stress at BBCH-51 and BBCH-55 in sunflower.


Introduction
Sunflower is an edible oilseed crop having short growing season, successfully grown under a range of water-limited environments globally [1][2][3]. It is a high yielding crop among edible oilseed crops; thus, could tighten up the production and consumption gap of edible oil in countries having huge edible oil import bill. Sunflower was introduced in Pakistan in 1960s as an oilseed crop and now recognized as top source of edible oil after cotton seed [4]. The supply of irrigation water is decreasing globally due to decreasing capacity and the annual inflow of water reservoirs [5]. Water availability usually remains insufficient and inconsistent in arid and semiarid climates. Therefore, low availability of irrigation water is a major limiting factor to attain the maximum yield potential of the field crops under such climates. The most of the cultivated areas in Pakistan are categorized as arid or semiarid, which face increasing shortage of freshwater availability that would decrease by 32% in 2050 [6]. Water shortage during growing season exerts severe negative impacts effects on crop production. The severity of these negative impacts varies greatly depending on the susceptibility of different growth and developmental stages. Soil water deficit restricts plant establishment, growth and development, which consequently ends with decreased crop productivity [7]. Water use efficiency is low in arid and semiarid areas due to high application and conveyance losses. Therefore, water use efficiency should be improved in these regions by appropriate agronomic measures. The timing of irrigation can significantly improve the water use efficiency in these regions [8]. Water use efficiency could be increased by using deficit or regulated deficit irrigation to achieve higher crop yield [9]. The accumulation of compatible solutes, e.g., proline, glycinebetaine and soluble sugars can enhance the capacity of field crops to acclimatize to drought stress. These solutes are highly soluble and low in molecular weight. Even higher cytosolic concentrations of these compatible solutes are not toxic for plants [10]. Compatible solutes detoxify reactive oxygen species produced under water deficiency, enhance membrane stability and protect enzymatic and protein structure, which collectively help plants to avoid cellular dehydration.
Under water deficient conditions, lower osmotic potential results from accumulation of compatible solutes which help plants in attracting water molecules into the cells and eventually cell turgor is maintained to relatively normal level [11]. The ABA is a vital phytohormone in crop plants which is involved in abiotic stress tolerance. Similarly, reduced water availability due to drought stress triggers ABA biosynthesis in plant tissue [12]. The ABA is subsequently translocate to the guard cells, where it stimulates stomatal closure and ultimately improves the water relations of the plant through adaptive physiological expressions, including lowering transpiration and increasing influx of water into the roots. The ABA plays vital role during dormancy functions like germination, root architecture modulation, vegetative growth and seed development. Thus, ABA could ameliorate the negative consequences of water deficiency [13]. Several studies have reported that ABA improves plant water relations under drought stress and increases crop yield. The improvement in yield and plant water relations of sunflower by exogenously applied ABA has also been reported. However, there are limited reports on the role of different doses of ABA applied at BBCH-51 and BBCH-55 in improving plant water relations and yield. Therefore, this study was conducted to investigate the role of different doses of ABA in improving plant water relations and accumulation of compatible solutes under drought stress. Further, determining the sunflower growth stage which better responds to exogenous application of ABA was the other aim of the study.

Materials and Methods Description of experimental site and seed collection
This two-year field study was conducted at Agronomic Research Farm, University of Agriculture, Faisalabad, Pakistan. The study area is characterized as semiarid [14]. The soil was sandy clay loam with 64.3, 17.8 and 17.9% sand silt and clay, respectively. The pH and EC of the soil were 8.14 and 1.39 dS m -1 , respectively. Similarly, the total organic matter, total nitrogen, available phosphorus and available potassium were, 0.58%, 0.59%, 7.31 ppm and 191 ppm, respectively. The meteorological data were obtained from the near weather station for the entire experimental period and presented in (Figure 1). The seeds of sunflower hybrid (Hysun-33) were obtained from Pakistan Oil Seed Development Board, Regional Office, Faisalabad.

Experimental treatments
Drought stress was imposed at BBCH-51 (bud initiation) and BBCH-55 (flower inititaiton) growth stages of sunflower by withholding irrigation (Table 1). A well-watered treatment where no irrigation was skipped was also included in the study. Similarly, ABA was applied at two different doses, (i.e., 5 and 10 µM) at the same groth stages where irrigation was skipped and no application of ABA was regarded as control. The experiment was laid out according to randomized complete block design with the factorial arrangement. Drought stress was the main factor, whereas ABA application was regarded as sub-factor. All treatments of the study had three replications and the experiment was repeated for two years.

Crop husbandry
Two cultivations, with tractor-mounted cultivator, followed by planking were applied to prepare seedbed suitable for plant growth. Seeds were planted on 3 rd week, i.e., 17 th and 12 th February during 1 st and 2 nd year of the study, respectively.
Seeds were sown on ridges with the help of dibbler by keeping seeds rate was kept 8 kg ha -1 . Ridges-to-ridge and plant-to-plant distances were kept 75 and 25 cm, respectively, which were maintained after thinning. Nitrogenous fertilizer as urea was applied at the rate of 150 kg N ha -1 in two equal splits, i.e., just before sowing and with first irrigation. Phosphatic fertilizer was added in the soil just before the sowing at the rate of 100 kg P 2 O 5 ha -1 as diammonium phosphate. Standard plant protection measures were applied to avoid any stress except described in the treatments.

Statistical analysis
Normality and homogeneity of variance in the collected data was tested first. Shapirowilk normality teste was used to assess the normality in the data and parameters with non-normal distribution were normalized by arcsine transformation technique. Homogeneity of variance was visually inspected by plotting the residuals before statistical analysis. The differences among the experimental years were tested by paired t test, which indicated significant differences. Therefore, the data of both years were analyzed, presented and interpreted separately. One-way analysis of variance (ANOVA) was then used to test the significance in the data for control, drought stress at BBCH-51 and drought stress at BBCH-55, separately. Least significance difference test at 5% probability was used to separate the treatment means where ANOVA indicated significant differences [19].

Results and Discussion Leaf water and osmotic potential (-MPa)
Drought stress at BBCH-51 and BBCH-55 significantly influenced leaf water potential (Ψ w ) and osmotic potential (Ψ s ) of sunflower ( Table 2). The ABA application under drought stress at at BBCH-51 and BBCH-55 variably affected Ψ w and Ψ s . Significant reduction in Ψ w and Ψ s was noted with ABA application at at BBCH-51 and BBCH-55 under wellwatered treatment of the study. ABA application to the plants exposed to water stress at BBCH-51 and BBCH-55 significantly increased Ψ w and Ψ s compared to the control. The lowest Ψ w and Ψ s were recorded under well-watered treatment, whereas the highest Ψ w and Ψ s were recorded for 5 and 10 µM ABA application at BBCH-51 and BBCH-55 (Table 2). Similar trend of Ψ w and Ψ s was recorded at BBCH-51 and BBCH-55 in both years of the study. Drought stress at BBCH-51 and BBCH-55 made the Ψ w more negative, whereas ABA application ameliorated the negative impacts to significant extent. The Ψ w was less negative with ABA application compared with no ABA application. The improvement in Ψ w through ABA application served as a proxy of improved tolerance ending with moisture conservation. The ABA accumulation under water-limited environments enables the plants to partially close stomata [20, 21] which result in reduced transpiration; hence, moisture is conserved. Drought stress either at BBCH-51 or BBCH-55 reduced Ψ w , which resulted in a parallel decrease in Ψ s . The accumulation of solutes in plant cells linked to osmotic adjustments was responsible for decreased Ψ s [22, 23]. The Ψ s became less negative with ABA application in the current study. A higher reduction in Ψ s was noted with ABA application at BBCH-51 compared with BBCH-55. The accumulation of compatible solutes e.g., GB, Pro, and TTS helped the plants to improve Ψ s under drought stress. The accumulation of these solutes detoxify reactive oxygen species, stabilize membranes and enzymatic structures ultimately hinder dehydration [24-26]. Leaf turgor pressure (MPa) Turgor pressure (Ψ p ) was significantly reduced by drought stress imposed at BBCH-51 and BBCH-55, and the reduction was higher under drought stress at BBCH-55 during both years of the study. Exogenous ABA application decreased Ψ p under well-watered conditions, whereas improved Ψ p under drought stress imposed at BBCH-51 and BBCH-55 in each experimental year. The highest and the lowest Ψ p under wellwatered conditions was recorded with no ABA application and 10 µM ABA application at BBCH-55, respectively (Table 2). Similarly, the highest and the lowest Ψ p under drought stress at BBCH-51 was noted with 5 µM ABA application at BBCH-51 and no ABA application, respectively. Likewise, the highest and the lowest Ψ p under drought stress at BBCH-55 was noted with 5 µM ABA application at BBCH-55 and no ABA application, respectively (Table 2). Decreased Ψ s under droughty stress is regarded as major physiological adaptation for maintaining Ψ p . However, the activities of certain enzymes which cause starch breakdown and other substances resulting in negative Ψ s are increased by ABA application. The Ψ p was significantly decreased by drought stress at BBCH-51 and BBCH-55. The reduced Ψ p is directly related to decreased Ψ w in the current study [26, 27]. The application of ABA significantly improved Ψ p , and the improvement was higher with ABA application at BBCH-51 compared with BBCH-55. Conserved plant moisture due to stomatal closure was responsible for the improved Ψ p [28, 29].  (Table 2). Similarly, the highest and the lowest RWC under drought stress imposed at BBCH-51 were recorded with 5 µM ABA application at BBCH-51 and no application of ABA, respectively. In the same way, the highest and the lowest RWC under drought stress imposed at BBCH-55 were recorded with 5 µM ABA application at BBCH-55 and no application of ABA, respectively ( Achene yield (kg ha -1 ) Achene yield was significantly reduced by drought stress, and reduction was severe when drought was imposed at BBCH-55. The exogenous application of ABA decreased the achene yield under wellwatered conditions compared to no application of ABA. However, achene yield was improved under drought stress at BBCH-51 and BBCH-55 with ABA application compared with no ABA application ( Table 3). The highest and the lowest achene yield under stress-free conditions was recorded for no ABA application and 5 µM ABA application at BBCH-55, respectively. When drought stress was imposed at BBCH-51, the highest achene yield was noted with 5 µM ABA application at BBCH-51, whereas the lowest was recorded with 10 µM ABA application at BBCH-55. Likewise, the highest and the lowest achene yield was recorded with 5 µM ABA application at BBCH-55, whereas the lowest was recorded with 10 µM ABA application at BBCH-51 (Table 3). Drought stress significantly hampered the achene yield; however, ABA application helped to improve achene yield to significant extent. The reduction in achene yield was higher under drought stress at BBCH-55 compared to BBCH-51. The reduction in achene yield can be directly linked with disturbed plant water relations and growth. The ABA-mediated yield improvement can be explained with improved plant water relations and osmotic adjustments in the current study. The higher reduction in achene yield at BBCH-55 might be linked with pollen abortion as flowers were initiating at this stage. The application of 5 µM ABA either at BBCH-51 or BBCH-55 improved the achene yield compared with no application. However, higher dose, i.e., 10 µM lowered achene yield compared to control indicating that it might be toxic for plants.