untitled ACTA BOT. CROAT. 75 (1), 2016 31 Acta Bot. Croat. 75 (1), 31–38, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0018 ISSN 0365-0588 eISSN 1847-8476 Physiological responses of two halophytic grass species under drought stress environment Zamin Shaheed Siddiqui1*, Huda Shahid1, Jung-Il Cho2*, Sung-Han Park2, Tae-Hun Ryu2, Soo-Chul Park2 1 Stress Physiology Lab., Department of Botany, University of Karachi, Karachi – 75270, Pakistan 2 National Academy of Agricultural Sciences, Rural Development Administration, Suwon 441-707, Republic of Korea Abstract – The physiological responses of two halophytic grass species, Halopyrum mucronatum (L.) Staph. and Cenchrus ciliaris (L.), under drought stress were evaluated. Biomass accumulation, relative water con- tent, free proline, H2O2 content, stomatal conductance, photosynthetic performance and quantum yield (Fv/Fm ratio) were studied. Under drought conditions, these halophytic plants expressed differential responses to wa- ter defi cit. Stomatal conductance and free proline content were higher in H. mucronatum than in C. ciliaris, while H2O2 content in H. mucronatum was substantially lower than in C. ciliaris. Performance index showed considerable sensitivity to a water defi cit condition, more so in C. ciliaris than in H. mucronatum. Results were discussed in relation to comparative physiological performance and antioxidant enzymes activity of both halophytic grasses under drought stress. Keywords: antioxidant enzymes activity, Cenchrus ciliaris, chlorophyll a fl uorescence, drought stress, Halo- pyrum mucronatum, photosystem II * Corresponding author, e-mail: zaminss@uok.edu.pk, jungilcho@korea.kr Introduction Drought and salinity have long been known as the most prevalent abiotic stresses inhibiting the growth and produc- tivity of many wild and domestic plant species across the world (Qadir 2008, Naz et al. 2010). In locations with lim- ited water resources and an increasing human population, conventional crop production might not be able to meet food demands. In this distressing situation, effective mea- sures are adopted not only to minimize crop losses but also to fi nd alternate means of food production. The only eco- nomic solution considered in the present circumstance is the use of halophytic plants as an alternate source of food and therefore their growth performance should be tested in arid and saline habitats (Khan and Duke 2001, Nedjimi 2011). For that, focus on developing halophytes as cash crops in the future should be amplifi ed (Breckle 2009). In the sub-continent, the salt range and large coastal area enable a large number of halophytes or salt tolerant plant species to grow. Among them, Halopyrum mucrona- tum L. Staph. and Cenchrus ciliaris L. are important and widely occurring halophytic grasses. Most of these halo- phytic grasses have been investigated and physiological ex- planations regarding their salt tolerance have been provided (Khan and Ungar 1999, Saini et al. 2007, Siddiqui and Khan 2011). However, the effects of drought stress on these plants and their physiological mechanism have not been ex- amined. Generally, C. ciliaris L. is considered to be an important pasture grass and is being used for cattle and sheep produc- tion in arid and semiarid regions (Khan and Ungar 1999, Saini et al. 2007, Sidiqui and Khan 2011). H. mucronatum is an excellent salt-tolerant grass species, and is used as fodder (Siddiqui and Khan 2011). Furthermore, it was also reported that these two grasses might have the ability to tol- erate long dry seasons under varying soil conditions indi- cating some degree of drought tolerance (Ayerza 1981, De Leon 2004). It was reported that some of the metabolic reactions triggered by drought and salinity are similar. Among them, osmotic adjustment, changes in relative water content, maximum quantum yield and dry mass accumulations are well known (Munns et al. 2002, Kwon et al. 2009, Siddiqui et al. 2014). Early responses to drought or salt stress are generally the same, apart from acting as water stress either qualitatively (saline) or quantitatively (amount of water) and the specifi c ion effect. Therefore, it is hypothesized that those halophytic grasses that showed salt stress tolerance in saline habitats may also exhibit drought tolerance in dry envi ronments. Hence, the physiological performance and SIDDIQUI Z. S., SHAHID H., CHO J.-I., PARK S.-H., RYU T.-H., PARK S.-C. 32 ACTA BOT. CROAT. 75 (1), 2016 antioxidant enzymes activity of two known salt tolerant grasses in drought stress environment have been examined. Materials and methods Plant materials The two halophytic grass species: H. mucronatum (L.) Staph. and C. ciliaris L were used for experiments. Caryop- ses were collected at maturity from these plants growing on dunes of Hawks Bay beach, Karachi, Pakistan. They were collected on December 2013. Hulled seeds were cleaned and stored in a refrigerator prior to use. Germination, growth and treatments The seeds were surface sterilized in 0.52% sodium hy- pochlorite solution for one minute and rinsed thoroughly with sterilized distilled water. Seeds were pre-soaked in dis- tilled water for 4 h. Ten seeds were placed in 90 mm steril- ized Petri plate. Plants were allowed to grow in a growth chamber (Hotpack USA) at 25–28 ± 2 °C day / night tem- perature with 69–80% humidity. Light intensity varied be- tween 2000 and 2300 μmol m–2 s–1. After 20 days, equal- sized seedlings were then transferred to pots with a diameter of 30.48 cm. There were fi ve plantlets in each pot with 6 replications for each treatment, i.e., one for control and oth- er for the drought treatment. The plantlets were grown up to four leaf stage and then drought was induced up to 7 days when soil moisture content reached 15%. After 7 days, the stomatal conductance (gs) and chlorophyll fl uorescence was recorded on the youngest fully expanded leaf between 9:00 – 11:00 AM using a steady state diffusion porometer, Mod- el SC-1 (Decagon devices) and a chlorophyll fl uorescence meter (OS-30p+, Opti-Science, USA) respectively. After- wards, plants were harvested and biomass production, rela- tive water content, free proline quantifi cation, H2O2 content and antioxidant enzymes activity were examined. Chlorophyll fluorescence After half an hour of dark adaptation, the chlorophyll fluorescence parameters, minimal chlorophyll fl orescence (F0) and maximal fluorescence (Fm), were measured in or- der to determine the maximum quantum yield (Fv/Fm ratio) (Maxwell and Johnson 2000) of ten fully expanded leaves using a portable fluorometer, model OS-30p+ (Opti-Sci- ence, USA). Pigment analysis Leaf samples (500 mg) were ground in 10 mL of 96% methanol and then centrifuged at 4000 rpm for 10 min. To- tal chlorophyll (Chl a+b), chlorophyll a (Ca), chlorophyll b (Cb) and total carotenoid (Cx+c) contents were determined (Lichtenthaler 1987). The supernatant was separated and the absorbance was read at 666, 653 and 470 nm in UV – Vis Spectrophotometer (Shimadzu), respectively. Later the pigments were quantifi ed according to the following formu- las: Ca = 15.65 × A666 – 7.340 × A653 Cb = 27.05 × A653 – 11.21 × A666 Cx+c = 1000 × A470 – 2.860 × Ca – 129.2 × Cb/245 Free proline content Free proline content was estimated according to Bates et al. (1973). Fresh leaf samples (500 mg) were homoge- nized in 10 mL of sulphosalicylic acid (3% w/v). Later, the extract was fi ltered through Whatman No. 2 fi lter paper. To 2 mL of the aliquot, 2 mL of acid ninhydrin and 2 mL of glacial acetic acid were added and the contents were boiled at 100 °C for an hour. The mixture was further extracted with 2 mL of toluene by mixing thoroughly with vigorous stirring for 15 to 20 s. The upper layer was separated from the aqueous phase and absorbance was read at 520 nm against toluene blank. Hydrogen peroxide content Hydrogen peroxide (H2O2) was estimated by the proce- dure of Sergiev et al. (1997). Fresh leaf samples (500 mg) were homogenized in 5 mL 0.1% (w/v) trichloroacetic acid (TCA) using ice bath. Afterwards, the homogenate was centrifuged at 12,000 g for 15 min. To 0.5 mL supernatant, 0.5 mL of 10 mM potassium phosphate buffer and 1 mL of 1 M potassium iodide (KI) were added. The absorbance was read at 390 nm. The H2O2 contents were estimated us- ing a standard curve. Relative water content Four leaf strips of 4 cm2 were excised randomly and fresh weights (FW) were determined. For the measurement of turgid weight (TW), leaves were left in distilled water for 24 h under low irradiance conditions. Samples were then oven-dried at 80 °C for 48 h and dry weight (DW) was de- termined. Relative water content (RWC) was calculated ac- cording to Barrs and Weatherley (1962) according the for- mula: Relative water content = (FW – DW / TW – DW) × 100 Enzyme assays Leaf samples (500 mg) were randomly collected and crushed in liquid nitrogen at 4 °C and homogenized in 10 mL protein extraction buffer containing Tris-HCl pH 6.8, 50 mg polyvinylpyrrolidone, 0.05 mM ethylenediaminetet- raacetic acid (EDTA). The contents were centrifuged at 12,000 rpm in a refrigerated micro centrifuge (Smart R-17, Hanil) for 10 min. Total protein was estimated by the meth- od of Bradford (1976). Catalase (CAT; EC 1.11.1.6) activity was estimated by the method of Patterson et al. (1984). The decomposition of H2O2 was measured at 240 nm taking Δε as 43.6 mM cm–1. Reaction assay (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 addition of 0.1 mL enzyme extract at 25 °C. The decrease in absorbance at 240 nm was used to calculate the activity. One unit of CAT activity is defi ned as the amount of enzyme that catalyzes the conversion of 1 mM of H2O2 min–1 at 25 °C. HALOPHYTIC GRASSES HALOPYRUM AND CENCHRUS UNDER DROUGHT ACTA BOT. CROAT. 75 (1), 2016 33 Ascorbate peroxidase (APX; EC 1.11.1.11) activity was performed by the method of Nakano and Asada (1981). The reaction mixture (2.0 mL) contained 50 mM potassium phosphate buffer (pH 7.0), 0.2 mM EDTA, 0.5 mM ascor- bic acid and 0.25 mM H2O2. The reaction was started after the addition of 0.1 mL enzyme extract at 25 °C. The de- crease in absorbance at 290 nm for one minute was record- ed and the amount of ascorbate oxidized was calculated from the extinction coeffi cient 2.8 mM cm–1. The unit of activity is expressed as micromole of ascorbic acid oxi- dized min–1 at 25 °C. Superoxide dismutase (SOD; EC 1.15.1.1) activity was performed by the method of Beyer and Fridovich (1987). The reaction mixture consisted of 27.0 mL of 0.05 M potas- sium phosphate buffer (pH 7.8), 1.5 mL of L-methionine (300 mg per 2.7 mL), 1.0 mL of nitroblue tetrazolium salt (14.4 mg per 10 mL), and 0.75 mL of Triton X-100. Ali- quots (1.0 mL) of this mixture were delivered into small glass tubes, followed by the addition of 20 mL enzyme ex- tract and 10 mL of ribofl avin (4.4 mg per 100 mL). The cocktail was mixed and then illuminated for 15 minutes in an aluminium foil-lined box, containing 25 W fl uorescent tubes. In a control tube the sample was substituted for by 20 mL of buffer and the absorbance was measured at 560 nm. The reaction was stopped by switching off the light and placing the tubes in the dark. Increase in absorbance due to the formation of formazan was measured at 560 nm. Under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples were calculated by determining the percentage in- hibition per minute. One unit of SOD is the amount of en- zyme that causes a 50% inhibition of the rate for reduction of nitroblue tetrazolium salt under the conditions of the as- say. Statistical analysis All data from treated and control were subjected to sta- tistical analysis using SPSS 17.0 (IBM, USA). The values were expressed as mean and standard errors. t-test (p = 0.05) was computed between drought treatments and corre- sponding controls for each species separately. Levels of sig- nifi cance were expressed on bar graph with different letters. Results Drought stress signifi cantly reduced leaf fresh and dry mass in both halophytic grass species (Fig. 1). Fresh weight in both species was lower than in the control. However, de- crease in turgid weights was non-signifi cant within a spe- cies. On the other hand, dry weights in both the species were substantially lower under drought stress conditions than in the control. In comparison with their respective con- trols, a greater decrease was observed in H. mucronatum than in C. ciliaris. Subsequently, relative water content in leaves was slightly higher in H. mucronatum than in C. cili- aris in a drought stress environment. Halophytic grasses subjected to drought showed a de- crease in chlorophylls a and b and total chlorophyll that was signifi cant compared to control (Fig. 2). However, C. ciliaris showed higher decrease than H. mucronatum under drought stress conditions. Similarly, total carotenoid content de- creased in both species, although more so in C. ciliaris. 0.00 0.02 0.04 0.06 0.08 0.10 B io m as s (g ) 0.00 0.02 0.03 0.05 0.06 0.08 0.09 0.11 0.12 Control Drought B io m as s (g ) 0.00 0.01 0.02 0.03 0.04 0.05 R el at iv e w at er c on te nt ( % ) 0 20 40 60 80 100 Plant species H. mucronatum C. ciliaris H. mucronatum C. ciliaris a ab ab b a b b ab ab a a b a b b b FW TW DW C hl or op hy ll b ( μ g m g -1 F W ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Control Drought (6 d) T ot al c hl or op hy ll ( μ g m g- 1 F W ) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 T ot al c ar ot en oi ds ( μ g m g -1 F W ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 C hl or op hy ll a ( μ g m g -1 F W ) 0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10 Plant species H. mucronatum C. ciliaris H. mucronatum C. ciliaris a b a b a b a b a b a b a a a b Fig. 1. Biomass and relative water content of Halopyrum mucro- natum (L.) Staph. and Cenchrus ciliaris L. under drought stress. Bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separate- ly, according to paired “t” test at p < 0.05. Vertical lines on bar graphs represent mean ± standard error. FW – fresh weight, TW – turgid weight, DW – dry weight. Fig. 2. Total chlorophyll and carotenoid content of Halopyrum mucronatum (L.) Staph. and Cenchrus ciliaris L. under drought stress. Bars followed by the same letter denote no signifi cant dif- ference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05. Vertical lines on bar graphs represent mean ± standard error. SIDDIQUI Z. S., SHAHID H., CHO J.-I., PARK S.-H., RYU T.-H., PARK S.-C. 34 ACTA BOT. CROAT. 75 (1), 2016 A noticeable reduction was observed in performance in- dex (PIabs) and photochemical quenching (qP) in both the species under drought stress environment (Fig. 3). Howev- er, results showed that a higher reduction in PIabs and qP was recorded in C. ciliaris than in H. mucronatum under drought stress (Fig. 3). A marked reduction in stomatal con- ductance (gs) was observed in two tested halophytic grass species due to drought stress. However, reduction in stoma- tal conductance was greater in C. ciliaris than in H. mucro- natum. The level of drought stress damage was examined in terms of free proline and hydrogen peroxide (H2O2) produc- tion (Fig. 4). Leaf proline concentration of both grasses in- creased due to drought stress. However, H. mucronatum ac- cumulated more proline than C. ciliaris under drought stress condition. On the other hand, H 2 O 2 content in C. cili- aris increased signifi cantly as compared to H. mucronatum under stress. Activity of antioxidant enzymes like superoxide dis- mutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) was measured under drought stress against control and is illustrated in Fig. 5. Observations revealed that SOD and CAT activities of treated samples increased signifi cant- ly in H. mucronatum as compared to C. ciliaris. However, antioxidant activities of all tested enzymes were higher in treated samples of both halophytes compared to control. Among the treated samples H. mucronatum showed an in- crease that was substantial in SOD and CAT as compared to C. ciliaris Discussion The physiological responses of two halophytic grass H. mucronatum (L.) Staph., and C. ciliaris L., were evaluated under drought conditions. It was observed that biomass production was reduced in the two tested species under drought stress. It is well known that drought stress condi- tions cause substantial reduction in biomass and growth in many plant species (Mahiwal and Sutaria 1992, Ashraf et F v/ F m r at io 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 P er fo rm an ce in de x (P I a bs ) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Control Drought P ho to ch em ic al q ue nc hi ng ( q ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 S to m at al c on du ct an ce , g s (m m ol m -2 s- 1 ) 0 40 80 120 160 200 240 280 320 Plant species H. mucronatum C. ciliaris H. mucronatum C. ciliaris a a a a a b a b a a a a a b a b H 2 O 2 ( m m ol g -1 F W ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Control Drought Plant species F re e ro li ne c on te nt ( μ m ol g -1 F W ) 0 5 10 15 20 25 30 H. mucronatum C. ciliaris H. mucronatum C. ciliaris a b a b a a a b APX 0 1 2 3 4 5 6 Control CAT Sp ec if ic a ct iv ity (U ni t m g- 1 o f p ro te in ) 0 1 2 3 4 5 6 H. mucronatum C.ciliaris 0 50 100 150 Plant species SOD a b a b a b a b a b a b Fig. 3. Maximum quantum yield (Fv/Fm ratio), performance index (PIabs) and photochemical quenching (qP), and stomatal conduc- tance (gs) of Halopyrum mucronatum (L.) Staph. and Cenchrus ciliaris L. under drought stress. Bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05 level. Vertical lines on bar graphs represent mean ± SE. Fig. 4. Total free proline and H2O2 content of Halopyrum mucro- natum (L.) Staph. and Cenchrus ciliaris L. under drought stress. Bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separate- ly, according to paired “t” test at p < 0.05 level. Vertical lines on bar graphs represent mean ± SE. Fig. 5. Antioxidant enzyme activity of Halopyrum mucronatum (L.) Staph. and Cenchrus ciliaris L. under drought stress. Bars fol- lowed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, accord- ing to paired “t” test at p < 0.05 level. Vertical lines on bar graphs represent mean ± SE. HALOPHYTIC GRASSES HALOPYRUM AND CENCHRUS UNDER DROUGHT ACTA BOT. CROAT. 75 (1), 2016 35 al. 1998, Karsten and MacAdam 2001, Tavakol and Pakniy- at 2007, Siddiqui 2013). In the present study, RWC of both the halophytic species was signifi cantly reduced under drought conditions compared to control (unstressed) plants. However, the reduction of RWC in C. ciliaris was higher than that in H. mucronatum. Hence, it can be suggested that H. mucronatum has a better drought tolerance through the maintenance of higher water content in leaf under drought. Furthermore, it was observed that RWC in crop plants and the tolerance of the plants to stress are directly related (Schonfeld et al. 1988, Merah 2001, Siddiqui et al. 2014). Therefore, it may be suggested that H. mucronatum may have a better ability to regulate intracellular water relations through biomass accumulation than C. ciliaris under drought stress conditions. It is well documented that decline in RWC is related to cell membrane properties and its adaptability to environmental changes such as drought (Katerji et al. 1997, El Hafi d et al. 1998, De Pereira-Neto et al. 1999, Liu et al. 2002, Molnar et al. 2002, Blokhina et al. 2003). However, spatial differences among the species can- not be ruled out as water relation characteristics refl ect the physiological differences among species and cultivars. Nevertheless, RWC is a good indicator of drought tolerance or adaptation in various plant species (Ashraf et al. 1994, Siddiqui et al. 2014). Photosynthetic performances of the two halophytic grasses under drought conditions were examined in terms of their components, such as maximum quantum yield (Fv/ Fm ratio), photochemical quenching (qP), performance in- dex and photosynthetic pigments analysis. Drought stress caused a signifi cant reduction in total chlorophyll and caro- tenoid content. However, a higher decrease was observed in C. ciliaris as compared to H. mucronatum. Photosynthetic pigments like chlorophylls and carotenoids are responsible for converting energy and/or trapping it in chemical forms for almost all green plants. It was observed that plant me- tabolism is clearly linked with photosynthetic pigment and adversely affected by abiotic stress like drought (Li et al. 2012, Siddiqui et al. 2013, Reza and Hassan 2014). The de- crease in chlorophyll under water stress is primarily a result of injury in chloroplasts caused by reactive oxygen species, which are usually elevated as a consequence of drought (Smirnoff 1995, Siddiqui et al. 2014). In this study the per- formance index and quenching substantially were reduced from the maximum yield in both the species under drought stress. Observations showed that greater reductions in sto- matal conductance, PIabs and qP, were recorded in C. ciliaris than in H. mucronatum under drought. The Fv/Fm ratio, characterizes the maximal effi ciency yield of excitation en- ergy captured by “open” photosystem II reaction centres. This suggests that the photosynthetic activity of C. ciliaris might be decreased due to inhibition in chlorophyll synthe- sis and their quenching ability which may have an effect on the performance index (Lutts et al. 1996, Tijen and Ismail 2006, Siddiqui 2013, Siddiqui et al. 2014). Photosystem II (PSII) in photosynthetic response is related to chlorophyll and carotenoids concentration and it was varied against fl uctuating environmental (Baker 1991). Therefore, chang- es in photosynthesis under water stress conditions are to be expected. Likewise, performance index (PIabs) is an excel- lent indicator that showed plant fi tness and provides useful quantitative information about photosynthetic apparatus (Strauss et al. 2003, Xia et al. 2004, Oukarroum et al. 2007, Mehta et al. 2010, Stefanov et al. 2011). Likewise, photo- synthetic pigments and maximum quantum yield are impor- tant physiological parameters refl ecting the photosynthetic ability of plants in stressful environments. (Colom and Vaz- zana 2002, Parida et al. 2003, Waseem et al. 2006, Siddiqui et al. 2008). H. mucronatum accumulated more proline and less hy- drogen peroxide content than C. ciliaris. A greater accumu- lation of proline in response to drought stress is well docu- mented in many plants and maintains homeostasis in leaf (Abdel-Nasser and Abdel-Aal 2002, Parida et al. 2007, Sla- ma et al. 2007, Mostajeran and Rahimi-Eichi 2009, Kumar et al. 2011). It was suggested that an amino acid like proline might play a highly valuable role in plants exposed to vari- ous stress conditions. Besides acting as an excellent osmo- lyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a sig- nalling molecule, which results in substantial reduction in ROS activity (Hayat et al. 2012). Three possibilities can be predicted from the results: (1) H. mucronatum may have a certain chloroplast protein against oxidative damages, (2) high carotenoid concentra- tions and proline enhance the antioxidant ability of the H. mucronatum (3) the increased chlorophyll in H. mucrona- tum as compared to C. cilliaris provides a continuous and substantial energy supply to maintain quantum yield in drought stress. It has been suggested that the sensitivity, tol- erance, and response timing of plants to drought vary among species. For example, slow-growing species have been found to be more sensitive than fast-growing species (Waseem et al. 2006, Munns 2002). This was seen in water stress; some drought-tolerant plants developed fi tness by reducing leaf area and stomatal conductance to transpira- tion (Nativ et al. 1999, Ares et al. 2000). Thus, plants might adapt physiologically to drought conditions by reducing stomatal conductance to water vapour, increasing their wa- ter-use effi ciency (WUE). Tolerant plants have been ob- served to adapt two different strategies during drought: long-living annuals and perennials decrease their leaf size and/or stomatal conductance (Geber and Dawson 1997, Querejeta et al. 2003), while shorter-living annuals maxi- mize fi tness by increasing stomatal conductance (decreas- ing WUE) to increase carbon gain and avoid drought stress. This strategy lets them grow rapidly, fl ower early, and in- crease yield before the start of substantial soil drying (Ge- ber and Dawson 1997, Mckay et al. 2003). When drought is experienced at later developmental stages, selection should favour decreased stomatal conductance (high WUE) and smaller leaves, whereas when plants experience drought at early developmental stages, increased stomatal conduct- ance (low WUE) should be selected for and leaf size may be of no adaptive value. It was observed that antioxidant enzyme activity like SOD and CAT were higher in H. mucronatum than in C. ciliaris in a drought stress environment However, compared to control, both halophytes showed substantial increases in SIDDIQUI Z. S., SHAHID H., CHO J.-I., PARK S.-H., RYU T.-H., PARK S.-C. 36 ACTA BOT. CROAT. 75 (1), 2016 antioxidant enzymes activities. It was reported that antioxi- dant enzymes like SOD, CAT, APX and GR played a sig- nifi cant role in combating drought stress and maintaining substantial growth rate under stress (Siddiqui 2013, Vujčić and Radić Brkanac 2014). It was observed that under stress, two different defensive mechanisms are provoked: a) an antioxidant non-enzymatic system such as a synthesis of osmolytes and phenols, and b) antioxidant enzyme systems such as synthesis and activity of enzymes like SOD, APX, CAT etc. (Siddiqui et al. 2014) In conclusion, through higher photosynthetic perform- ance, photo-quenching and lower stomatal conductance, maintaining substantial higher relative water content, H. mucronatum may be considered to have more drought tol- erance than C. ciliaris. Furthermore, it was concluded that H. mucronatum maintains a substantially better perform- ance index and lower H2O2 contents than C. ciliaris, which may be due to greater antioxidant enzyme activity, such as SOD and CAT. Acknowledgments This work was supported by a grant from the Next-Gen- eration Biogreen 21 Program (PJ01131902 and PJ01123204), Rural Development Administration, Suwon, Republic of Korea. 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