36 ACTA BOT. CROAT. 77 (1), 2018 Acta Bot. Croat. 77 (1), 36–44, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0002 ISSN 0365-0588 eISSN 1847-8476 Physiological performance of sunflower genotypes under combined salt and drought stress environment Muhammad Umar, Zamin Shaheed Siddiqui* Stress Physiology Lab., Department of Botany, University of Karachi, Karachi – 75270, Pakistan Abstract – The physiological performance of some sunflower genotypes (S.28111, SF0049, Hysun-33, Hysun-39) under salt, drought stress separately and in combination was examined. Salt, drought and a combination of these stresses were applied to plants by gradual increments. The plants were exposed to stress for two weeks. Relative water content, osmotic potential, stomatal conductance, performance index, dark adapted quantum yield and chlorophyll contents were reduced upon salinity and drought stresses. However, when plants were subjected to a combination of these stresses, a greater reduction in all tested attributes was observed. Proline and carotenoid contents in drought stress were elevated compared to salt stress. Superoxide dismutase (SOD) and catalase (CAT) showed the highest ac- tivity in individual salt and drought stress with less accumulation of H2O2. Combined stress reduced the activity of antioxidant enzymes which ultimately decreased the physiological performance of sunflower plants. However, among the tested genotypes, S.28111 and SF0049 were found to be more tolerant to drought, salt and combined stress than both Hysun genotypes. The physiological performance of genotypes against salinity and drought individually and in combination is discussed in detail. Key words: combined stress, genotypes, photosynthesis, screening, single stress, sunflower * Corresponding author, e-mail: zaminss@uok.edu.pk Introduction The sunflower (Helianthus annuus) is the most impor- tant source of edible oil and fourth largest oilseed crop in the world and its production is still expanding. It is planted over an area of 319,743 hectares producing 420,487 tons annually in Pakistan (Amanullah and Khan 2011). Sunflower is mod- erately tolerant to water stress, but its growth and production are limited in drought and salt stress environments (Aziz et al. 2013). The growth and yield of sunflower are adversely affected by abiotic stress and its yield is reduced up to 60% (Mazahery-Laghab et al. 2003). It was observed that the gen- eral effects of salt and drought stress on plant are restricted to separate exposure of these stresses. However, physiological assessment of sunflower genotypes in a combination of salt and drought stress is rather scarce. Further, the effect of com- bined stresses on sunflower genotypes and the basic physio- logical and biochemical mechanisms are still unclear. There- fore, it is necessary to study the physiological performance of sunflower plants in conditions of combined salt and drought stresses. Further, a selection of sunflower cultivars with better physiological responses to combined abiotic stresses would provide novel options to growers under different environ- mental conditions, as in nature plants are exposed to a com- bination of several stresses at a time. In the last few decades drastic climatic changes have been observed, probably due to global warming, which has brought about not only a serious environmental threat but also sub- stantial reductions in the yield and the crop quality. Plants are frequently exposed to many extremes such as drought stress, low/high temperature, salt stress, flooding stress, and heavy metal toxicity while growing in nature. Among these, drought and salinity are the major environmental constraints that lead to detrimental effects on a plant’s life and hence crop produc- tivity. About 33% of the world’s arable land has to face crop re- ductions due to cyclic or unpredictable drought, whereas sa- linity, a huge and worldwide problem, has affected about one billion hectares of land (Wicke et al. 2011). Salt and drought mailto:zaminss@uok.edu.pk PLANT RESPONSE AGAINST COMBINED STRESS ACTA BOT. CROAT. 77 (1), 2018 37 stresses affect many morphological features and alter the physiological processes that are associated with plant growth and development (Siddiqui et al. 2008). It was reported that in drought stress, plant growth retardation is linked with pho- tosynthesis, respiration, and nutrient metabolism. Further, production of reactive oxygen species (ROS) in extreme en- vironments like high salt concentration adversely affects plant growth and development (Munns and Tester 2008). To improve agricultural productivity, it is necessary to develop resistant varieties by an understanding of the phys- iological and biochemical mechanisms or selecting better genotypes capable of performing well in stress conditions. It was reported that changes undergone by plants as a result of combined stresses are markedly different to responses to individual stresses (Grzesiak et al. 2016). Different plant spe- cies and even genotypes may have varying responses to salt and drought stresses. Therefore, the objectives of the pres- ent study were: 1) to assess the physiological performance of sunflower genotypes in salt and drought stresses separately or in combination, and 2) to identify a suitable genotype for a stress environment on the basis of its physiological and an- tioxidant performance. In this study, physiological and biochemical parameters used in the screening of salt or drought stress were selected (Siddiqui et al. 2016) and their correlations are presented. This approach may be useful to identify physiologically tol- erant genotypes of sunflower under water limited, salinity prone and combined stress environments. Materials and methods Plant material and experimental operation The experiment was carried out in a greenhouse at the Department of Botany, University of Karachi, Pakistan. Plas- tic pots (15 × 18 cm) were filled with 1.5 kg of air dried soil. The soil types used in this trial were sandy loam, pH 7.5. Seeds of Helianthus annuus (sunflower cultivars: S.28111, Hysun-33, Hysun-39, and SF0049) were collected from the seed certification department of the government of Pakistan. S.28111 and SF0049 are new sunflower genotypes originated by Arysta Life Science and FMC Corporation. Physiological performance of these genotypes under stress environment was not available. However, genotypes Hysun-33 and Hy- sun-39 were known to moderately tolerate drought and salt without the physiological explanation for this having been reported. Seeds were surface sterilized with 2% sodium hy- pochlorite for five minutes. Five seeds per pot were sown and 12 days after sowing (DAS) pots were thinned to three seed- lings per each pot. Salinity and drought treatments were ini- tiated 30 DAS and lasted till about 44 DAS. Control plants were irrigated with tap water and soil was kept humid (more that 70% water holding capacity) until harvesting. For salin- ity, 175 mM NaCl concentration (almost 17 dS m–1) was ap- plied in gradual increments. To achieve the desired salt con- centration, aliquots of the following NaCl solutions were used in order: 75 mM, 50 mM and 50 mM (Tab. 1). Later, soil was kept humid, retaining more than 70% water holding capac- ity throughout the experimental periods (Table 1). Drought stress was imposed by withholding water until soil moisture content (SMC) reached 20% and then maintained through- out the experiment. Combined salt and drought stress was achieved by progressive imposition of 175 mM NaCl i.e. 1st salt application was 75 mM, 2nd 50 mM and 3rd 50 mM and then withholding water until SMC fell to 20%. Soil moisture, electrical conductivity and pH were measured using soil sen- sor (SDI-12 hydra probe II, Stevens water, USA) and pH me- ter (Adwa AD111, Romania), respectively (Tab. 1). The ex- periment was arranged in a randomized block design with four replicates for each treatment. At the end of the experi- ment, physiological measurements, biochemical quantifica- tion, and phenotypic characters were studied. Chlorophyll fluorescence and stomatal conductance The stomatal conductance (gs) and chlorophyll fluores- cence were recorded on the youngest fully expanded leaf be- tween 9:00 AM – 11:00 AM using a steady state diffusion porometer, Model SC-1 (Decagon devices) and chlorophyll fluorescence meter (OS-30p+, Opti-Science, USA) respective- ly. For chlorophyll fluorescence leaves were dark-adapted us- ing clips. After half an hour of dark adaptation, the chlorophyll fluorescence parameters ratio of variable florescence to maxi- mum florescence (Fv/Fm), which reflected maximum photo- chemical efficiency of PS-II, and photosynthetic performance index (PIabs) were recorded (Maxwell and Johnson 2000). Af- terwards, plants were harvested and biomass production, rel- ative water content, free proline quantification, H2O2 content and antioxidant enzymes activity were examined. Photosynthetic pigments Fresh leaf samples (500 mg) were used for the extraction of pigments in 10 mL of 96% methanol, which was then centri- Tab. 1. Soil parameters: soil moisture contents (SMC) and electri- cal conductivity (EC) during gradual increment of the soil salinity/ drought treatments. Drought stress was imposed by withholding water. Parameters of the last treatment were maintained until the time of harvesting. ± SE indicates the standard error of mean. Bold values represent the final parameters values of each treatment at the time of harvesting. Stress type Salt application NaCl (mM) Day SMC (%) EC (dS m–1) pH Salinity 1st 75 1st 80–90 7.4±0.08a 7.98 2nd 50 3rd 80–90 12.0±0.15b 8.04 3rd 50 5th 80–90 16.9±0.05c 8.09 Drought -- -- 2nd 60–65 1.8±0.014a 8.01 -- -- 4rd 35–40 2.0±0.013ab 8.08 -- -- 6th 20–25 2.3±0.034b 8.13 Salinity + drought 1st 75 1st 80–90 7.4±0.08a 8.01 2nd 50 3rd 80–90 12.0±0.15b 8.05 3rd 50 5th 80–90 16.9±0.05c 8.11 -- -- 7th 60–70 19.4±0.08d 8.18 -- -- 9th 40–50 21.1±0.12e 8.19 -- -- 12th 20–25 24.2±0.17f 8.24 UMAR M., SIDDIQUI Z. S. 38 ACTA BOT. CROAT. 77 (1), 2018 fuged at 4000 rpm for 10 min. Total chlorophyll, chlorophyll a, chlorophyll b and carotenoid contents were determined ac- cording to Lichtenthaler (1987). The absorbance was read at 666, 653 and 470 nm using a spectrophotometer. The amounts of these pigments were calculated and expressed in µg mg–1 fresh weight (FW) (Lichtenthaler and Wellburn 1985): Ca = 15.65 × A666 – 7.340 × A653 Cb = 27.05 × A653 – 11.21 × A666 Carotenoids = (1000 × A470 – 2.860 × Ca – 129.2 × Cb) / 245 Where A = absorbance, Ca = chlorophyll a content and Cb = chlorophyll b content Leaf water status Leaf water status was evaluated as the leaf relative wa- ter content (RWC). RWC was determined from the young- est fully expanded leaves. The leaves were weighed immedi- ately after harvesting to obtain the fresh weight (FW). The same leaves were then subsequently rehydrated in distilled water for 4 h to obtain the turgid weight (TW), and finally dried for 48 h at 72 °C to obtain the dry weight (DW) (Barrs and Weatherley 1962). RWC was calculated by the following formula with slight modification and expressed in percent (Smart and Bingham 1974): RWC = FW – DW / TW – DW × 100 Osmotic potential (ψs) Three randomly selected fully developed leaves of each treatment and control were taken and frozen in liquid nitro- gen to measure ψs. The frozen leaves were thawed at room temperature and centrifuged at 12,000 g. Finally the osmolar- ity of extracted sap was recorded using an osmometer type 6 (Loser Messtechnik, Berlin, Germany) and expressed in bars. Hydrogen peroxide (H2O2) content H2O2 content was measured according to the procedure of Velikova et al. (2000). 100 mg of fresh leaf samples were ho- mogenized with 3 mL of 0.1% (w/v) trichloroacetic acid and then centrifuged at 12,000 g for 15 minutes. Later, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide were added to 0.5 mL of the supernatant. Finally, the absorbance was recorded at 390 nm. The amount of H2O2 was calculated using extinction coefficient (43.6 mM cm–1) and expressed as µmol g–1 FW. Proline analysis Proline was estimated according to the method of Bates et al. (1973). Fresh leaf samples (500 mg) were homogenized in 10 mL of sulphosalicylic acid (3% w/v) and the extract was filtered through Whatman’s No. 2 filter paper. In a 2 mL ali- quot, 2 mL of acid ninhydrin and 2 mL of glacial acetic acid were added and the contents were boiled for 1 h at 100 °C in a water bath. The reaction mixture was further extracted with 2 mL of toluene by mixing thoroughly with vigorous stirring for 15 to 20 sec. Chromophore containing toluene was sepa- rated from the aqueous phase. Later absorbance was recorded at 520 nm against toluene blank. Proline content in sample was estimated by referring to a standard curve made from known concentrations of proline using the following formula: µmoles proline g–1 FW = {(µg proline / ml × ml toluene) / 115.5 µg / µmol} / (g sample) / 5 Enzyme extraction 500 mg of leaf samples were collected and crushed in liq- uid nitrogen and then homogenized at 4 °C in 10 mL pro- tein extraction buffer containing 100 mM Tris-HCl (pH 6.8), 50 mg polyvinylpyrrolidone (PVP), and 0.1 mM EDTA. The contents were centrifuged at 14095 ×g for 15 min using Smart R-17, Hanil centrifuge. Total protein was estimated by the method of Bradford (1976). Catalase (CAT) activity was estimated by the method of Patterson et al. (1984). The decomposition of H2O2 was mea- sured at 240 nm taking the optical density at 240 nm as 43.6 mM cm–1 (extinction coefficient). Reaction mixture (3.0 mL) consisted of 10.5 mM H2O2 in 0.05 M potassium phosphate buffer (pH 7.0) and the reaction was initiated after the addi- tion of 0.1 mL enzyme extract at 25 °C. The decrease in ab- sorbance at 240 nm was used to calculate the activity. One unit of CAT activity is defined as the amount of enzyme that catalyzes the conversion of 1 mM of H2O2 min–1 at 25 °C. The assay for superoxide dismutase (SOD) activity was performed by following the method of Beyer and Fridrovich (1987). The reaction mixture (27.0 mL) with pH 7.8 was pre- pared using 23.75 mL of potassium phosphate buffer (0.05 M), 1.5 mL of L-methionine (300 mg per 2.7 mL), 1.0 mL of nitroblue tertrazolium salt (14.4 mg per 10 ml), 0.75 mL of triton X-100. One mL aliquots of assay mixture were trans- ferred into small glass tubes, followed by the 20 µL enzyme extract and 10 µL of riboflavin (4.4 mg per 100 mL) were added. The cocktail was mixed and then illuminated for 15 minutes in an aluminum foil-lined box, containing 25 W flu- orescent tubes. In a control tube the sample was replaced by 20 µL of buffer and the absorbance was measured at 560 nm. The reaction was stopped by switching off the light and plac- ing the tubes in the dark. Increase in absorbance due to for- mation of formazan was measured at 560 nm. Under the de- scribed conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples was calculated by determining the percentage inhibition per min- ute. One unit of SOD is the amount of enzyme that causes a 50% inhibition of the rate for reduction of nitro blue tetrazo- lium salt under the conditions of the assay. Statistical analysis Statistical analysis of the collected data was performed using the Duncan (1955) multiple range test (p ≤ 0.05) and Pearson’s correlation with the help of the personal computer software packages IBM SPSS Statistics (version 20). To test the differences among mean values, Duncan’s test was com- puted and the resultant values were expressed in a bar graph as alphabets. Difference in means and their F-values were cal- culated and are given in Table 2. PLANT RESPONSE AGAINST COMBINED STRESS ACTA BOT. CROAT. 77 (1), 2018 39 Results The application of single stress (drought and salt separate- ly) and combined stress showed significant reduction in bio- mass production of each tested sunflower genotype. Relative water content was substantially decreased in all treated geno- types under single and combined stress environment (Fig. 1). However, in combined stress, 45, 56, 49 and 37% reduction were observed in S.28111, Husun-33, Hysun-39 and SF0049, respectively (Tab. 2). Leaf area of each plant was decreased in all treatments compared to control. Reduction in leaf area was recorded up to 58, 59, 67 and 51% under combined stress in S.28111, Hysun-33, Hysun-39 SF0049, respectively (Tab. 2). Reduction in leaf number was the highest in Hysun-33, whereas the lowest reduction was observed in SF0049 (Fig. 1). Salt and drought stress significantly reduced the height of all sunflower genotypes in comparison to corresponding con- trols. It was observed that S.28111 is the least affected among the genotypes (Fig. 1). Higher reduction in shoot height was observed in Hysun-33 and Hysun-39, of about 42 and 38% as compared to their controls, respectively. Free proline produc- tion was significantly affected in the tested sunflower geno- types. It was observed that proline concentrations were high- er in combined stress as compared to drought or salt stress (Fig. 1). The order of proline accumulation in genotypes un- der combined stress varied for instance from the maximum in Hysun-39>Hysun-33>S.28111>SF0049 (minimum). Sunflower genotypes exhibited a substantial decrease in PIabs in all stresses as compared to control. Combined stress significantly reduced the PIabs in all the genotypes as com- pared to control and individual stresses (Fig. 2). However, the highest decreased in PIabs was shown in Hysun-33 under Tab. 2. Effects of double stress on performance index (PIabs), quantum yield (Fv/Fm ratio), stomatal conductance (gs), relative water con- tent (RWC), total chlorophyll content (T.chl), leaf area (LA), osmotic potential (ψs), catalase activity (CAT), hydrogen peroxide (H2O2), photochemical quenching (qP), carotenoids contents (Car), superoxide dismutase activity (SOD) in sunflower genotypes expressed as increment or decline percentage over control. Highest increment/decline is presented in bold. * denotes p-value <0.01, ** denotes p-value <0.001, ***denotes p-value <0.0001, ns – not significant. Decline % (–) Genotypes PIabs F-value Fv/Fm F-value gs F-value RWC F-value T.chl F-value LA F-value S.28111 31.8 16.4** 10.6 20.5*** 80.4 225*** 44.9 42.8*** 12.4 20.2*** 58.4 188*** Hysun-33 52.1 20.6** 21.0 35.2*** 75.0 110** 55.6 30.9** 63.1 1169* 59.2 202*** Hysun-39 32.8 14.3** 13.8 40.2*** 76.9 252* 49.6 125*** 32.8 58.0*** 67.4 74.4*** SF0049 21.8 11.6* 07.4 15.7** 77.8 452*** 37.2 13** 31.1 32.7*** 54.4 239*** Increment % (+) Genotypes ψs F-value CAT F-value H2O2 F-value qP F-value Car F-value SOD F-value S.28111 81.8 393** 50 38.4*** 43.2 109** 80.4 1.43ns 10.6 149*** 20.4 26.3*** Hysun-33 70.5 171*** 25 13.2** 48.9 83.8*** 75.6 15.8** 22.9 70.2*** 26.6 21.7*** Hysun-39 70.9 112** 40.3 25.0*** 49.2 57.9*** 79.4 0.33 ns 06.4 40.5* 21.2 18.5* SF0049 81.9 210*** 45 20.7*** 44.6 99.5** 34.1 5.88* 20.2 19.6** 17.1 18.8** Fig. 1. Effects of salt (S), drought (D) and combined (S + D) stress on shoot height, number of leaves, relative water content and free proline content of sunflower genotypes. Bar represents mean ± SE (n = 4). Same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. UMAR M., SIDDIQUI Z. S. 40 ACTA BOT. CROAT. 77 (1), 2018 combined stress. A marginal decline was detected in maxi- mum photochemical efficiency of photosystem II (Fv/Fm ra- tio) when sunflower plants were treated alone with salt and drought stress. A significant reduction was observed in Fv/Fm ratio under combined stress, of 21% in Hysun-33 whereas on- ly a 7.4% reduction was found in SF0049. In both individual and combined stresses photochemical quenching (qP) was not significantly affected. The stomatal conductance of each genotype was reduced by the salt and drought stress (Fig. 2). The stomatal conductance under salt and drought stress was higher in SF0049 than in the other genotypes. It was observed that both Hysun genotypes had the lowest stomatal conduc- tance in the individual stress conditions (Fig. 2). The plants showed a substantial reduction in total chloro- phyll content in stress environments (Fig. 3). Greater reduc- tion was found in Husun-33 in combined stress conditions than in the other genotypes. The total carotenoid concentra- tion was increased in stressful environment when genotypes were treated with single or combined stresses. The highest ca- rotenoid concentration was shown in Hysun-33 under com- bined stress whereas the lowest concentration was recorded in S.28111. Osmotic potential (ψs) of each genotype decreased in drought and salt stressed plants as compared to unstressed (control) plants. A greater decline in osmotic potential was observed during combined stresses (Fig. 4). Antioxidant en- zymes like SOD and CAT were examined in genotypes when plants were exposed to a single (drought/salt) and to com- bined (salt + drought) stresses. Plants treated with drought, salinity or in combination, showed significant increases in SOD and CAT as compared to control. It was observed that Fig. 2. Effects of salt, drought and combined (S + D) stress on Fv/Fm ratio, PIabs, qP and gs of sunflower genotypes. Values are mean ± SE (n = 4). Same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. Fig. 3. Effects of salt, drought and combined (S + D) stress on contents of photosynthetic pigments of sunflower genotypes. Values are mean ± SE (n = 4). Same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. PLANT RESPONSE AGAINST COMBINED STRESS ACTA BOT. CROAT. 77 (1), 2018 41 CAT and SOD activities were increased in all genotypes un- der single stress as compared to control (Fig. 5). More anti- oxidant activities were observed in S.28111 and SF0049 than in Hysun-33 and Hysun-39; however, SOD activity did not significantly differ in single stresses, drought or salinity. H2O2 is a known stress indicator in plants and its concentration in- creases in abiotic stress. The data showed that the concentra- tion of H2O2 increased in all genotypes under both drought and salt stress. However, H2O2 contents increased much more under drought stress than under salt stress, concentrations getting higher still under combined stress. It was observed that production of H2O2 contents were reduced in S.28111 and SF0049 more than in other genotypes. Table 2 showed that both Hysun genotypes had the highest decline in RWC, leaf area, total chlorophyll and PIabs under combined stress compared to their control, whereas S.28111 and SF0049 had the highest osmotic potential and CAT activity. The outcome of correlation analysis is given in Table 3. Proline, carotenoids and H2O2 are negatively correlated with all the parameters and are positive with each other. Catalase activity was negatively correlated with PIabs, Fv/Fm ratio, RWC, gs, and chlorophyll content. The correlation of CAT is found to be significant with gs, carotenoid, H2O2, ψs and SOD. PIabs significantly correlated with SOD and non-significantly with CAT whereas gs was non-significantly correlated with CAT and significantly with SOD. Discussion Reduction in plant biomass in response to drought and salt stress is a common phenomenon. Substantial reductions in number of leaves and height of plants were recorded in Hy- sun-33 and Hysun-39, more so than in S.28111 and SF0049. RWC of each sunflower genotype was reduced in both single Fig. 4. Effects of salt, drought and combined (S + D) stress on activity of antioxidant enzymes CAT and SOD (expressed as unites mg–1 proteins), H2O2 content and osmotic potential of sunflower genotypes. Values are mean ± SE (n = 4). Same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. Tab. 3. Pearson correlation among performance index (PIabs), maximum quantum yield (Fv/Fm ratio), relative water contents (RWC), stomatal conductance (gs), leaf area (LA), total chlorophyll content (T.chl), carotenoids content (Car), proline, hydrogen peroxide (H2O2), osmotic po- tential (ψs), and superoxide dismutase (SOD). ** denotes p<0.01 (2-tailed), * denotes p<0.05 level (2-tailed), ns – not significant. PIabs FvFm RWC gs LA T.chl Car Proline H2O2 ψs SOD CAT PIabs 1 .735** .886** .700** .742** .594* –.654** –.836** –.847** .763** –.520* –.413 ns Fv/Fm 1 .820** .748** .830** .822** –.630** –.890** –.840** .529* –.160 ns –.279 ns RWC 1 .841** .857** .599* –.606* –.942** –.929** .718** –.395 ns –.465 ns gs 1 .920** .598* –.516* –.850** –.910** .777** –.474 ns –.611* LA 1 .609* –.583* –.918** –.912** .712** –.441 ns –.589* T-Chl 1 –.715** –.640** –.712** .529* –.224 ns –.297 ns Car 1 .551* .666** –.627** .413 ns .612* Proline 1 .918** –.673** .340 ns .391 ns H2O2 1 –.846** .533* .611* ψs 1 –.629* –.604* SOD 1 .830** CAT 1 UMAR M., SIDDIQUI Z. S. 42 ACTA BOT. CROAT. 77 (1), 2018 stress and combined stress. However, maximum reduction was observed in Hysun-33 and minimum reduction found in SF0049 and S.28111. RWC is a better indicator of leaf water status and is successfully used to determine stress resistance or tolerance in many crop plants (Siddiqui et al. 2014). Many reports reveal that RWC is reduced under drought and salin- ity (Masoumi et al. 2010) but those plants that maintain high RWC under extreme stress are supposed to be more stress tol- erant. Hence it is assumed that sunflower genotypes SF0049 and S.28111, which maintained substantial water in leaf un- der combined stress, could be more stress tolerant than other genotypes. It is suggested that reduced shoot height, leaf area and number of leaves in sensitive genotypes may be due to their leaves having less RWC. The accumulation of proline in a single stress and a combined stress environment was ob- served. S.28111 accumulated a sufficient amount of proline under a combined stress environment. The accumulation of compatible osmolytes such as proline has been regarded as a basic strategy for the protection and survival of plants under abiotic stress (Alia et al. 2001). The better plant height, num- ber of leaves, leaf area and RWC in S.28111 under combined stress could be due to the higher accumulation of proline. Relative water content and proline content are negatively cor- related (r = ‒0.942, Table 3). Therefore, it may be suggested that an increase in proline concentration might be due to the reduction in RWC under osmotic stress, indicating that syn- thesis of proline became higher as soon as RWC declined. Results show that Hysun-33 had the highest decline in Fv/Fm (21%) and PIabs (53%) of all the genotypes, while SF 0049 showed better results having 7.4% decline in Fv/Fm ra- tio and 31% in PIabs in a single stressor in a combination (Tab. 2). Chlorophyll fluorescence is a sensitive indicator that of- ten fluctuates in stress environments (Weng et al. 2008). In stress sensitive plants, Fv/Fm ratio decreases under limited wa- ter conditions indicating chronic photo-inhibition (Zlatev 2009). The vitality of the plant could be characterized by the PIabs, which is more sensitive to abiotic stresses (Oukarroum et al. 2007). Combined stress significantly reduced the PIabs in all the genotypes as compared to control and single stresses. However, the highest decrease in PIabs was shown in Hysun-33 under combined stress reflecting poor functionality of both PSI and PSII under stress (Strasser et al. 2004). A very small decline was observed in SF0049 under S + D as compared to control indicating better performance under stress. The PIabs showed significant correlation with all the tested param- eters except catalase. RWC were highly correlated with PIabs (0.886). This correlation showed that the reduction in perfor- mance index might be due to the decreased RWC. The sto- matal conductance of each sunflower genotype was reduced by the salt and drought stress. The maximum reduction in stomatal conductance was observed in S.28111 and SF0049 under combined stress. During salt and drought stress, less water is available for the plants to facilitate their metabolism smoothly. In osmot- ic stress, an imbalance between water uptake by roots and water loss by transpiration causes the plant to wilt (Lafitte 2002). Therefore, water use efficiency may be an important strategy to increase fitness under osmotic stress environment. Reports have shown that in some stress tolerant genotypes ABA is synthesized and accumulated which leads to stoma- tal closure thus avoiding unnecessary transpiration to main- tain substantial cellular osmotic potential. Drought tolerant plants maximize fitness by decreasing stomatal conductance in response to the shortage of water (Ares et al. 2000). The genotypes S.28111 and SF0049 showed 80 and 78% decline in stomatal conductance under combined stress compared to control, respectively (Tab. 2). It is suggested that these two genotypes tolerate salt and drought by decreasing stomatal conductance and thereby increasing water use efficiency. Lev- els of plant sensitivity, tolerance, and response timing against water stress fluctuate among genotypes / species. For exam- ple, slow-growing plant species have been found to be more sensitive than fast-growing species (Munns 2002). It was ob- served that tolerant plants adapt two different strategies dur- ing abiotic stress: long-living annuals and perennials decrease their leaf size and/or stomatal conductance (Siddiqui et al. 2014), while shorter-living annuals maximize fitness by in- creasing stomatal conductance (lessening water-use efficien- cy) to increase carbon gain and avoid stress. This tactic lets them grow speedily, flower early and increase yield before the start of substantial soil drying (McKay et al. 2003). Thus, it is thought genotypes S.28111 and SF0049 might adopt the early developmental strategy and enhance stress tolerance whereas both Hysun genotypes adopt the late developmental strate- gy and thereby have less tolerance to salt, drought and com- bined stress. Genotype S.28111 showed the highest chlorophyll con- tents in a combined stress environment of all the tested gen- otypes. Further, abiotic stress causes ROS production that damages the chloroplast and as a result a reduction in chlo- rophyll contents occur (Smirnoff 1995). Reduction in chlo- rophyll under drought and salinity stresses has been reported in many plant species including sunflower (Manivannan et al. 2007). Higher reduction in chlorophyll contents in Hysun genotypes might be due to imbalance in ROS production and scavenging mechanism. The non-enzymatic antioxidants like carotenoids and proline contents were negatively correlated with photosynthetic parameters and their correlation with PIabs, Fv/Fm and total chlorophyll were highly significant. This negative correlation might be due to the protective strategy of plants under harmful environmental conditions in which chlorophyll contents are reduced and carotenoids increased. Sunflower plants treated with drought and salinity sin- gly or in combination, showed a significant increase in SOD and CAT compared to control. It was observed that CAT and SOD activities were increased under stresses compared to control but combined stress reduced the activity of both enzymes in all the genotypes. However, more antioxidant ac- tivities were observed in S.28111 and SF0049 than in Hy- sun-33 and Hysun-39. It has been reported that plant toler- ance can be maintained by controlling the ROS production through non-enzymatic mechanisms, including carotenoids, proline and phenolic compounds (Jaleel et al. 2009) or to pre- vent plants from oxidative damage by antioxidant enzymes PLANT RESPONSE AGAINST COMBINED STRESS ACTA BOT. CROAT. 77 (1), 2018 43 activity like CAT and SOD (Quan et al. 2008). The present study showed that CAT and SOD activities of sunflower gen- otypes were negatively correlated with H2O2 concentrations. It is presumed that the lower H2O2 production in drought stress may be due to enzyme activities or it may be related to higher carotenoid synthesis, which may lower H2O2 produc- tion in chloroplasts, thus avoiding oxidative damages. CAT is one of the enzymes that detoxify H2O2 in plants and it is mostly located in peroxisomes. CAT use H2O2 as a substrate and changes it to H2O and O2 as products. It is clear that S.28111 and SF0049 genotypes showed better photosynthetic performance than the other genotypes under combined stress (Tab. 2). Both genotypes showed minimum decline in Fv/Fm ratio, performance index and total chlorophyll. This minimum decrease under combined stress environment showed that S.28111 and SF0049 showed better physiological performance than either of the Hysun genotypes. The maxi- mum increment in ψs and CAT activity and minimum incre- ment in H2O2 contents also showed that S.28111 and SF0049 have better tolerance than the Hysun genotypes in individu- al and combined stress. The parameters used in this trial are significantly correlated (Tab. 3). Despite combined salt and drought stress these parameters are useful to identify genotypes tolerant to water-limited and salinity-prone environments. The present study has shown that S.28111 and SF0049 exhibited a greater degree of tolerance than the Hysuns in terms of better physiological performance against salinity, drought and their combination. This might be due to their early development strategy, as compared to both Hysun gen- otypes. It was based on the evaluation of genotypes respond- ing to stress tolerance, which is shown as percentage increase (+) /decrease (–) over control and illustrated in Table 2. The genotypes S.28111 and SF0049 showed the lowest decline in PIabs, Fv/Fm ratio, RWC and chlorophyll content. However, in all treatments the highest increase in osmotic potential, proline contents, qP, carotenoids contents and antioxidant enzymes activities was recorded. Results of the present in- vestigation showed that SF0049 and S.28111 genotypes have better tolerance than either of the Hysun genotypes. Gen- otypes S.28111 and SF0049 not only showed better photo- synthetic performance but also reduced the concentration of H2O2 in all stresses. Therefore, it may be suggested that gen- otypes S.28111 and SF0049 could be used in field trials for a more comprehensive study. However, more detailed molec- ular work is needed to provide more convincing arguments. Acknowledgements We are grateful to the department of Botany of the Uni- versity of Karachi for supporting the study and the seed manager of FMC Corporation (Pakistan), and Seed Cer- tification Department of Pakistan for providing seeds. The authors also acknowledge financial support from NCGC South Korea. References Jaleel, C. A., Manivannan, P., Wahid, A., Farooq, M., Al-Juburi, H. J., Somasundaram, R., Panneerselvam, R., 2009: Drought stress in plants: a review on morphological characteristics and pigments composition. International Journal of Agriculture and Biology 11, 100–105. Lafitte, R., 2002: Relationship between leaf relative water content during reproductive stage water deficit and grain formation in rice. Field Crops Research 76, 165–174. Lichtenthaler, H. K., 1987: Chlorophylls and carotenoids: pig- ments of photosynthetic membranes. Methods in Enzymol- ogy 148, 350–382. Lichtenthaler, H. K., Wellburn, A. R., 1985: Determination of total carotenoids and chlorophylls a and b of leaf in different sol- vents. Biochemical Society Transactions 11, 591–592. Manivannan, P., Abdul Jaleel, C., Sankar, B., Kishorekumar, A., Somasundaram, R., Lakshmanan, G. M. A., Panneerselvam, R., 2007: Growth, biochemical modifications and proline me- tabolism in Helianthus annuus L. as induced by drought stress. Colloids and Surfaces B: Biointerfaces 59, 141–149. Masoumi, A., Kafi, M., Khazaei, H., Davari, K., 2010: Effect of drought stress on water status, elecrolyte leakage and enzy- matic antioxidants of kochia (Kochia scoparia) under saline condition. Pakistan Journal of Botany 42, 3517–3524. Maxwell, K., Johnson, G., 2000: Chlorophyll fluorescence – a prac- tical guide. Journal of Experimental Botany 51, 659–668. Mazahery-Laghab, H., Nouri, F., Abianeh, H. Z., 2003: Effects of the reduction of drought stress using supplementary irrigation for sunflower (Helianthus annuus) in dry farming conditions. Pa- jouheshva-Sazandegi, Agronomy and Horticulture 59, 81–86. Mckay, J. K., Richards, J. H., Mitchell-Olds, T., 2003: Genetics of drought adaptation in Arabidopsis thaliana. I. Pleiotropy con- Alia, J. M., Mohanty, P., Matysik, J., 2001: Effect of proline on the production of singlet oxygen. Amino Acids 21, 195–200. Amanullah, Khan M. W., 2011: Interactive effect of potassium and phosphorus on grain quality and profitability of sunflower in northwest Pakistan. Pedosphere 21, 532–538. Ares, A., Fownes, J. H, Sun, W., 2000: Genetic differentiation of in- trinsic water-use efficiency in the Hawaiian native Acacia koa. International Journal of Plant Sciences 161, 909–915. Aziz, R., Shahbaz, M., Ashraf, M., 2013: Influence of foliar appli- cation of triacontanol on growth attributes, gas exchange and chlorophyll fluorescence in sunflower (Helianthus annuus L.) under saline stress. Pakistan Journal of Botany 45, 1913–1918. Barrs, H. D., Weatherley, P. E., 1962: A re-examination of the rela- tive turgidity technique for estimating water deficits in leaves. Australian Journal of Biological Sciences 15, 413–428. Bates, L. S., Waldren, R.P., Teare, L. D., 1973: Rapid determination of free proline water stress studies. Plant and Soil 39, 205–207. Beyer, W. F., Fridovich, I., 1987: Assaying for superoxide dis- mutase activity: some large consequences of minor changes in condition. Analytical Biochemistry 161, 559–566. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Duncan, D. B., 1955: Multiple range and Multiple F-test. Biomet- rics 11, 1– 42. Grzesiak, T. M., Janowiak, F., Szczyrek, P., Kaczanowska, K., Os- trowska, A., Rut, G., Hura, T., Rzepka, A., Grzesiak, S., 2016: Impact of soil compaction stress combined with drought or water logging on physiological and biochemical markers in two maize hybrids. Acta Physiologiae Plantarum 38, 109. http://www.sciencedirect.com/science/journal/00032697 http://www.sciencedirect.com/science/journal/00032697 UMAR M., SIDDIQUI Z. S. 44 ACTA BOT. CROAT. 77 (1), 2018 tributes to genetic correlations among ecological traits. Mo- lecular Ecology 12, 1137–1151. Munns, R., 2002: Comparative physiology of salt and water stress. Plant Cell and Environment 25, 239–250. Munns, R., Tester, M., 2008: Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681. Oukarroum, A., El-Madidi, S., Schansker, G., Strasser, R. J., 2007: Probing the responses of barley cultivars (Hordeum vulgare L.) by chlorophyll fluorescence OLKJIP under drought stress and rewatering. Environmental and Experimental Botany 60, 438–446. Patterson, B., Macrae, E., Ferguson, I., 1984: Estimation of hydro- gen peroxide in plant extracts using titanium (IV). Annals of Clinical Biochemistry 139, 487–492. Quan, L., Zhang, B., Shi, W., Li, H., 2008: Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species net- work. Journal of Integrative Plant Biology 50, 2–18. Siddiqui, Z. S., Cho, J. l., Park, S. H., Kwon, T. R., Ahn B. O., Lee, K. S., Jeong, M. J., Kim, K. W., Lee S. K., Park S. C., 2014: Physiological mechanism of drought tolerance in transgenic rice plants expressing Capsicum annuum Methionine sulfox- ide reductase B2 (CaMsrB2) gene. Acta Physiologiae Planta- rum 36, 1143–1153. Siddiqui, Z. S., Khan, M. A., Kim, B. G., Huang, J.S., Kwon, T. R., 2008: Physiological responses of Brassica napus genotypes to combined drought and salt stress. Plant Stress, Global Sci- ence Books. Siddiqui, ZS., Shahid, H., Cho, J. I., Park, S. H., Ryu, T. H., Park, S. C., 2016: Physiological responses of two halophytic grass spe- cies under drought stress environment. Acta Botanica Cro- atica 75, 31–38. Smart, R. E., Bingham, G. E., 1974: Rapid estimates of relative wa- ter content. Plant Physiology 53, 258–260. Smirnoff, N., 1995: Antioxidant systems and plant response to the environment. In: Smirnoff, N. (ed.), Environment and plant metabolism: flexibility and acclimation, 217–243. Bios Sci- entific Oxford. Strasser, R. J., Tsimilli-Michael, M., Srivastava, A., 2004: Analy- sis of the fluorescence transient. In: George C., Papageorgiou C., Govindjee (eds.), Chlorophyll fluorescence: a signature of photosynthesis, 321–362. Advances in Photosynthesis and Respiration Series. Springer, Dordrecht. Velikova, V., Yordanov, I., Edreva, A., 2000: Oxidative stress and some antioxidant system in acid rain-treated bean plants. Pro- tective role of exogenous polyamines. Plant Science 151, 59–66. Weng, X. Y., XU, H. X., Yang, Y., Peng, H. H., 2008: Water-water cycle involved in dissipation of excess photon energy in phos- phorus deficient rice leaves. Biologia Plantarum 52, 307–313. Wicke, B., Smeets, E., Dornburg, V., Vashev, B., Gaiser, T., Turken- burg, W., Faaij, A., 2011: The global technical and economic potential of bioenergy from salt-affected soils. Energy and En- vironmental Science 4, 2669–2681. Zlatev, Z., 2009: Drought-induced changes in chlorophyll fluores- cence of young wheat plants. Biotechnology & Biotechnologi- cal Equipment. 23/2009/SE. https://www.researchgate.net/journal/0006-3134_Biologia_Plantarum