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South African Journal of Botany 105 (2016) 226–233

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South African Journal of Botany

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Effects of exogenous salicylic acid on Impatiens walleriana L. grown
in vitro under polyethylene glycol-imposed drought
D. Antonić, S. Milošević ⁎, A. Cingel, M. Lojić, M. Trifunović-Momčilov, M. Petrić, A. Subotić, A. Simonović
Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bul. despota Stefana 142, 11060 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: We describe the responses of Impatiens walleriana to polyethylene glycol (PEG)-induced physiological drought
Received 3 November 2015 and the potential of exogenous salicylic acid (SA) as stress-ameliorating agent. Impatiens shoot culture was
Received in revised form 28 March 2016 established on 16 different media containing 0–3% PEG and 0–3 mM SA. After prolonged drought (60 days),
Accepted 5 April 2016
water relation parameters, oxidative stress indicators, and growth responses of the shoots to PEG and/or SA
Available online 19 April 2016
were recorded. PEG reduced growth, fresh weight, the number of developed leaves and shoots (proliferation
Edited by J Van Staden rate, PR), relative water content, and chlorophyll content. PEG increased leaf water loss (LWL) and caused accu-
mulation of proline, H2O2, and malondialdehyde. The activities of catalase, superoxide dismutase, and peroxidase
Keywords: were increased in response to PEG in a dose-dependent manner, with specific peroxidase isoforms induced by
Drought drought. Exogenous SA counteracted the effects of PEG on growth, physiological and biochemical parameters,
Impatiens walleriana except on proline accumulation. SA was particularly effective in enhancing PR, preserving LWL, and protecting
Polyethylene glycol photosynthetic pigments and membranes from oxidative damage. Proline accumulation was strongly enhanced
Reactive oxygen species by both PEG and SA. SA had differential effects on different peroxidase isoforms. SA may be safely used in 2–3 mM
Salicylic acid
concentration for drought protection of Impatiens with no negative effects.
Water stress
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction (Burnett et al., 2005). However, physiological and biochemical
responses to water stress in Impatiens have not been studied.
Genus Impatiens (Balsamiaceae) includes over 900 species of annual Drought is a major abiotic stress that affects growth, nutrient rela-
or perennial herbs (Grey-Wilson, 1980). Due to their beauty and long tions, photosynthesis, assimilate partitioning, and respiration (Farooq
flowering period, many Impatiens species are cultivated worldwide as et al., 2009). Common consequences of drought stress are reduction of
bedding or potted plants. I. walleriana is the most popular among the transpiration rate, relative water content (RWC), and leaf water poten-
Impatiens species, having fleshy, succulent leaves and a variety of flower tial. In order to cope with water deficit, plant cells can decrease their
colors. The major problem in production, transport, and sale display of osmotic potential and thus maintain turgor by accumulation of compat-
Impatiens is related to its tendency to quickly wilt when drought- ible solutes, primarily proline (Farooq et al., 2009; Hayat et al., 2012).
stressed. Prolonged water deficit in potted I. walleriana plants reduced Proline (Pro) is not only involved in the osmotic adjustment of plant
height, shoot number, dry weight, and flower number (Blanusa, cells but may also stabilize cell membranes and scavenge reactive
2009), while osmotic stress in hydroponically grown I. walleriana re- xygen species (ROS) (Hayat et al., 2012).
duced height and width of the plantlets, as well as their root length One of the most devastating consequences of drought is the onset of
oxidative stress imposed by imbalance between ROS production and the
capacity of enzymatic and non-enzymatic antioxidative defense sys-
tems (Farooq et al., 2009). Water stress damages photosynthetic appa-
Abbreviations: CAT, Catalase; CDPKs, Calcium dependent protein kinases; DW, Dry ratus and impairs electron transfer in chloroplasts and other cellular
weight; FW, Fresh weight; LWL, Leaf water loss; MDA, Malondialdehyde; MS, Murashige
compartments, resulting in the accumulation of ROS—superoxide
and Skoog medium; PEG, Polyethylene glycol; POX, Peroxidase; PR, Proliferation rate;
Pro, Prolin; ROS, Reactive oxygen species; RWC, Relative water content; SA, Salicylic anion radicals (O2 −), hydroxyl radicals (•OH), hydrogen peroxide
acid; SABPs, Salicylic acid binding proteins; SOD, Superoxide dismutase; TW, Turgid (H2O2), and singlet oxygen (1O2) (Hayat et al., 2008; Demiralay et al.,
weight. 2013). ROS may react with proteins, membrane lipids, and other cellular
⁎ Corresponding author. Tel.: +381 11 2078 393; fax: +381 11 2078 404. components, causing oxidative damage (Farooq et al., 2009; Demiralay
E-mail addresses: dragana.antonic@ibiss.bg.ac.rs (D. Antonić), snezana@ibiss.bg.ac.rs
(S. Milošević), cingel@ibiss.bg.ac.rs (A. Cingel), milena.lojic@ibiss.bg.ac.rs (M. Lojić),
et al., 2013). One of the products of membrane lipids peroxidation is
milanag@ibiss.bg.ac.rs (M. Trifunović-Momčilov), marija.nikolic@ibiss.bg.ac.rs (M. Petrić), malondialdehyde (MDA), used as a reliable indicator of ROS formation
heroina@ibiss.bg.ac.rs (A. Subotić), ana.simonovic@ibiss.bg.ac.rs (A. Simonović). and membrane damage (Kadioglu et al., 2011; Bidabadi et al., 2012;

http://dx.doi.org/10.1016/j.sajb.2016.04.002
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Life Sciences. (NBT) chloride. 2-thiobarbituric acid. plant responses to exogenous SA depend on Pro standard) was mixed with 50 μl ninhydrin reagent (0.7). and nized in liquid nitrogen with 1 ml of 0. and SOD activities were expressed as samples for all analyses were also collected from in vitro cultured shoots μmol min−1 mg−1 of soluble protein (U mg−1). Maryland.11. Hayat et al. and stress level under aseptic conditions. 2009.2. 2014.35% ethanol the species. Piwowarczyk et al.2 (STSC Inc. where W2 is leaf weight 1.. 20. shoot explants (10–12 mm long) were excised from the seed.. according to ANOVA using the StatGraphics software version 4. The absorbance of the pigments was measured with UV- culture allows for convenient but stringent control of the physical envi. EC (%) is defined as ratio (FW − W2)/FW × 100.. and 7 gl− 1 agar. was carried out as 10% commercial bleach (5% sodium hypochlorite). 2004)..15. are commonly conducted with polyethylene glycol (PEG) used to simu. under different treatments after 60 days. 0. 2012). all of which may result in oxidative stress (Farooq et al. and involved in various biotic interactions.2-dihydroxyindane-1. and 664 nm. only leaves with petioles were used. and corrected for endogenous SA level (Horvath et al. malondialdehyde. All experiments were repeated three times. Johnsons) were surface sterilized in for CAT and POX activities and in-gel POX assay.000g at 4 °C for 10 min and the water potential of the culture media (Van den Berg and Zeng.. CAT. the formula given by Barrs and Weatherley (1962): RWC (%) = Rockville. addition to culture 4 min. 2012. foliar solution). from 250 mg leaf tissue by grinding in liquid nitrogen followed by ex- es. PEG is neutral. 2. Quantification of pigments. Free amino acids were extracted resulting in enhanced tolerance toward different types of abiotic stress. (2000). 260 mM L-methionine.. as the amount of enzyme where the NBT reduction (to blue formazan) eters such as main shoot length. irrigating. 2014). with or without SA (0–3 mM).3-dione) to produce in low concentrations commonly has an acclimation-like effect.3.1).1. Miura and Tada. and ratio was 50%. and growth parameters Extraction of total soluble proteins. EC 1. NBT photoreduction was measured using microplate the number of shoots (proliferation rate. For all biochemical analyses.. visible spectrophotometer (Agilent 8453. 15. a yellow compound (Friedman. Tissue used as a control. H2O2.5. after evaporation (Xing et al. 2012. such as drought (Farooq et al. Miura and Tada. DW is dry weight recorded of P b 0. Statistical analyses (LVL) were determined for whole shoots. catalase (CAT. while RWC and leaf water loss 2. 2013). The absorbance of the media or hydroponic solution). determined by subjecting shoots to rehydration for 2 h. Demiralay et al. The Adeniyi.5 ml of 20% trichloroacetic acid in 0. The cell debris was spun et al. non-penetrating. or 25 μl of crude tions of PEG8000 (0–3%). (1987). at irradiance of (Tesla Pančevo. 10. Materials and methods 2. prepared in parallel for each sample. However. inert.8). 2012).. Bidabadi et al. Piwowarczyk et al. and variable stresses including 2.1% trichloroacetic acid. / South African Journal of Botany 105 (2016) 226–233 227 Alam et al. seed soaking. we present physiological and biochemical responses of in vitro ninhydrin. the leaf samples (100 mg) were homoge- late drought. After reaction mixture contained 100 mM potassium phosphate buffer (pH 30 days. EC 1.. One SOD unit was described 47 μmol m−2 s−1 at the culturing surface. The Pro grown Impatiens to PEG-simulated drought and exogenous SA. Statistical differences among experimental treatments were assessed by RWC was determined after 60 days in culture. Plants grown under field or greenhouse conditions may be simulta- neously exposed to multiple.. 100 mgl− 1 myo-inositol. 2015). and proline drought. D. down at 14. separated and evaporated to dryness. The cultures protein extract.. The most important antioxidative enzymes that are after drying the samples at 75 °C for at least 24 h. with five plants used for 2. The ho- osmotically active polymer that induces dehydration by decreasing mogenate was centrifuged at 15.05. Plant material. Here. The in vitro studies of drought responses H2O2 content was determined as described by Velikova et al. USA).000g and the supernatant was mixed with 500 μl chloro- enous SA can improve growth under water deficit conditions in a num. USA) at 470. Growth param. form and 750 μl HPLC-grade water. Pérez-Clemente and Gómez-Cadenas. The mean differences were compared by (FW − DW)/(TW − DW) × 100. exog. which is primarily due to increased antioxidative capacity (Horvath traction in 500 μl HPLC-grade methanol. supernatant was mixed with 0. excessive light or heat.04 mM riboflavin. 2014). The samples were incubated in water bath at 100 °C for spraying. 2013).1. amount was determined from the standard curve.. The reaction mixture was kept under fluorescent light were grown under long day (16/8 h photoperiod). . 2014). (2012). 7. developmental stage.000g at 4 °C for 10 min.11.. 2013. 2007. mode of SA application (e. Enzymatic assays 2.1. The concentrations of chlorophyll a and b and caroten- close monitoring of plant growth and physiological responses to stress oids were calculated using the equations proposed by Lichtenthaler (Sakthivelu et al. In addition. PR) and leaves per plant were reader at 540 nm. since 2010. and clarified by centrifugation at in plants and involved in diverse physiological and developmental pro. POX. and placed described by Milošević et al.1.. stem injection.. 65 W) for 60 min at 25 °C. LWL control the H2O2 level in cells—superoxide dismutase (SOD. 2004). 2010. Total chlorophyll and carotenoids were extracted from leaves using 2009.4. and peroxidase (POX. MDA was then determined spectrophoto- cesses including plant pathogen defense responses and responses to metrically as described by Health and Packer (1968). and 20 μl of sample (or et al. salinity. Odjegba and extracted with chloroform. and 5. as blanks. Hayat et al. at 25 ± 2 °C.g. 2013. 1. and TW is turgor commonly induced in response to oxidative stress are enzymes that weight. Determination of relative water content and leaf water loss each treatment (n = 15) and the results are expressed as means ± SE. 2008. ronment. as well as spectrometric assays I.. 15. and 648. cooled on ice. 1962) containing slightly modified method described by Beyer and Fridovich (1987). Application of SA Pro reacts with ninhydrin (2. cooled and diluted with 930 μl ethanol. SOD activity was determined by aseptically on MS medium (Murashige and Skoog. A 30 gl−1 sucrose. abiotic stresses. The aqueous phase was re- ber of species (Hayat et al. where FW is fresh weight of shoots.. Free Pro was determined by “non-classical” ninhydrin reaction. 2012. Marcińska samples were resuspended in 125 μl water. The reaction mixture was heated at 95 °C for Salicylic acid (SA) is considered as potent phytohormone.. walleriana seeds (Busy Lizzie. nutrient supply. Alam et al. 2006. 2 mM EDTA. fresh and dry weight of the shoots. Antonić et al. Pérez-Clemente and Gómez-Cadenas. 2007. non-ionic. culture conditions. Bidabadi et al. 2008. Manohar et al.6).5 mM nitroblue tetrazolium lings and transferred to MS supplemented with increasing concentra. least significant difference (LSD) method with statistical significance measured at the end of the experiment. the applied SA concentration and the yellow reaction product was measured at 350 nm. For determination of MDA.5% Sakthivelu et al. complex. The mixtures without crude enzyme extract were determined on the 60th day of the PEG and/or SA treatment.. background absorbance using reactions with 50 μl ethanol instead of by. ubiquitous 30 min in water bath. rinsed. Plant tissue in vitro 96% ethanol.. 2008.

3. respectively 3 mM SA.2 and 2.9% CAT reduction in plants photosynthetic pigments content in I.2. 2c). Finally.7% lower PR. While low tent in the medium. 3% PEG. as indicators of oxidative stress. Fig. compared to the plants grown on (1–2% PEG) water stress up to 26. 1. application of 2–3 mM SA significantly reduced CAT activity both in Drought stress reduced the content of chlorophyll and total pig.1% lower than control (Fig. In unstressed plants treated with ments down to 51. when applied in drought conditions. while the (Fig. concentrations positively affected the average leaf number in plants cul. Increasing SA concentrations duced increment in H2O2 and MDA up to 27. and their FW was reduced to 61. concluded that SA completely reverses the effects of drought stress on ative effect of drought up to 26. so that shoots grown on 3 mM SA had LWL activity. All SA treatments treated with 2–3 mM SA was at the level of unstressed control.5 and 51.7% of the control values. in To investigate the effects PEG-induced drought on I. while SA ameliorates oxidative stress in effects on growth of the unstressed plants (Fig.1% and 33. walleriana and 3% PEG + 3 mM SA treatment the chlorophyll level was 2. When I. 2d).1% plants treated with 3% PEG and any of tested SA concentrations (Fig. . Bar = 1 cm. walleriana plants were cultured on media supplemented with increasing PEG concentration. SA slightly improved RWC in control and PEG-treated seed.1-fold more shoots per plant when treated 3. / South African Journal of Botany 105 (2016) 226–233 3. (Fig. Exogenously applied SA had no ber of leaves per plant in control and mildly stressed plants. Water stress caused a signifi- trol (Fig. The application of 2–3 mM SA significantly increased same SA concentration of 3 mM caused a 73.1% (Fig. 2a).2-fold higher chlorophyll and total pigments content. 3. Plants grown on 3% PEG were smaller in comparison to the control plants. 4c). The effect of PEG-imposed drought stress on ROS accumulation was Increasing PEG concentrations in the medium progressively reduced evaluated as H2O2 content in leaves of plantlets cultivated under differ- plant height. in comparison to the control.4%) only for 3 mM SA (Fig.3. Both PEG and SA enhance Pro accumulation in Impatiens with SA. enzymes.1. the than in 3% PEG treatment).1-fold higher in comparison to control (Fig. 3a). Results of shoots grown on 3 mM SA had 2. membrane polyunsaturated fatty acids. but the SA effect was statistically significant only in shoots ex- posed to intense stress. However. had 54. lower in comparison to the control) was recorded for shoots on 3% PEG (Fig. which was sta. induced by 1–2% PEG. Effect of PEG and SA on hydration level and photosynthetic pigments to 1.6-fold (Fig. all SA doses re- growth parameter up to 27.7-fold higher the potential of exogenous SA as a stress-ameliorating agent. plants growing on 3% PEG had drastically elevated CAT activity.9% as compared to tivated on 3% PEG. a decomposition product of the peroxidized leaves. SA treatment had no effect on height of the unstressed cant increase in H2O2 and MDA content in Impatiens leaves of up to plants. 1). growth effect of SA was observed on shoot development (PR). Effect of polyethylene glycol (PEG) and salicylic acid (SA) treatments on growth and morphology of in vitro cultivated Impatiens walleriana. 2b). Since the level of H2O2 and MDA in water-stressed plants tistically significant (14. but 2–3 mM SA extenuated the retarding effect of moderate 1. since the unstressed plants had up to 2.5. unstressed and in stressed plants. and the number of leaves and shoots per plant (Fig.g.4% lower than in control. so that leaves cultivated on 3% PEG. media without PEG (Fig. LWL increased up to 45. the most prominent these parameters. Pro content was significantly increased under all SA treatments. walleriana treated with 3% PEG and 3 mM SA grew better than plants treated with PEG alone.8% (Fig. ent conditions. The PEG-induced drought and Since water stress elevates ROS levels. the CAT activity was 33. so that lowest RWC (38. respectively. had very little effect on total CAT plants SA decreased LWL. 4a and b). PEG treatment causes. levels in untreated plants.5%.2% shorter. respectively. whereas in stressed plants this improvement was up to 4.4 and 2. it can be significantly increased FW of the stressed plants. thus ameliorating neg. but it even increased it above the control I. 3b).0% less termined as level of MDA. except that 3 mM SA reduced H2O2 content for 14. 4a and b). 85. Although SA had no in- content of in vitro grown Impatiens fluence on Pro accumulation in control plants. It RWC of the Impatiens shoots decreased with increasing PEG content seems that Pro accumulation reaches a plateau at ≈ 37 μg g−1 FW in in the medium in a dose-dependent manner.228 D. However. FW. The able to reverse the effects of PEG on this parameter.1% that of con. reducing the negative effect of drought on this control. in water stressed plants. respectively caused a slight increase in FW of the unstressed plants. 5a).7% (Fig. e. Exogenous SA ameliorates growth-retarding effects of PEG SA in higher (2–3 mM) concentrations was not only able to prevent chlorophyll and total pigment decline in water-stressed plants (e. Antonić et al. walleriana shoots were cultivated on MS media containing serial con. centrations of PEG and/or SA.6 times compared to the normal conditions. 2).g. were determined. but all SA significant effect on H2O2 and MDA accumulation in unstressed plants.23 times. plants I. walleriana leaves. whereas the level of lipid peroxidation in leaves was de- When exposed to 3% PEG. the activities of antioxidative exogenous SA had opposing effects on LWL: with increasing PEG con. Pro content in leaves increased up 3. plants were 50. 2). for 27. All concentrations of SA were which was 3.4.1%. SA did not affect the num. while in unstressed and mild stress. 2c). whereas 3 mM SA had no apparent 3. PEG and SA have opposing effects on antioxidative enzymatic activities lings. 3c and d).

Growth and developmental responses of I. Marcińska et al. 2012) and tomato (Hayat et al. but concomitantly inhibited isoforms A. mitochondria. physiological. wheat (Bajji et al. thus increasing electrolyte leakage and water loss wile at 3% PEG.. 2009). fresh shoot weight (c) and the number of shoots per plant or proliferation rate (PR) (d) were recorded on the 60th day of the culture on PEG and/or SA-containing media.. Changes in plant height (a). and other cellular and is the most meaningful index for dehydration tolerance (Alam components causing oxidative stress (Farooq et al. exogenous SA increased POX in unstressed plants Accumulation of Pro. metal chelation. 5b). is (for 34..) for the same PEG treatment are marked by asterisks (⁎P b 0. walleriana shoots cultivated on plasts. forms C... and ROS scavenging (Verbruggen and might have different effects on different POX isoforms. 2015).. 4a) and by increase in MDA content. 4c). observed in I.01. 2009). also in mustard (Alam et al. POX activity was nearly 4-fold higher as compared to (Hayat et al. RWC reflects plant water status damage proteins. walleriana Farooq et al. average number of leaves per plant (b).. but in some cases Drought also caused an increase in total POX activity. which is usual response of plants to water stress. 2009. ⁎⁎P b 0. as well as in higher in 3% PEG-treated plants than in control (Fig.1. reduced SOD activity in unstressed plants (for 34. 2013). LWL is somewhat ambig- the drought effect by lowering SOD for 25. and plasma membrane (Farooq PEG-containing media (Fig.4-fold banana (Bidabadi et al.. Pro is also involved in stabilization of mem- branes and proteins.. Data represent mean ± SE (n = 15).. Unlike RWC. antioxidation. 2000.. SA significantly Celosia argentea (Odjegba and Adeniyi. Lathyrus (Piwowarczyk et al. reported (Fig. 3b) and banana (Bidabadi et al. the same protein Hermans. 2000. 2009). 2008. Marcińska but particularly isoforms B and I. indicating .. 4. / South African Journal of Botany 105 (2016) 226–233 229 Fig. resulting in in- creased formation of ROS as by-products of electron transport in chloro- Reduction of RWC. which can explain the observed LWL increase.. 2013). and ⁎⁎⁎P b 0. all important grounds of drought tolerance—osmotic adjustment. 2013). and had a often correlated with drought tolerance. and other species. 4. POX was dramatically increased even at low stress (1% PEG). Farooq et al. decreased it in 3% PEG-treated plants (for 49. which may be increased in water-stressed plants. Impatiens (Fig. RWC was also significantly reduced in mustard (Alam treatment causes oxidative stress in Impatiens was confirmed both by et al.2% in plants treated with 3% uous parameter. et al. being 1. buffering cellular redox potential under stress. 2004). nucleic acids. walleriana to PEG-imposed drought and exogenous salicylic acid (SA). as in wheat (Xing et al. is one of the common consequences et al.. and biochemical changes in I. Indeed.. Discussion acting as a sink for carbon and nitrogen for use after stress relief. tomato (Hayat et al. Hayat et al. B. 2009. Antonić et al. 2008). 2013). the 3 mM SA treatment induced iso. membrane lipids. shoots cultivated on PEG-containing media Drought may disrupt the electron transport chains. 5c). wheat (Bajji et al.. Developmental. 2013). Drought may cause mem- and SOD. while 3% PEG caused induction of all POX isoforms.. H. 2012) seedlings exposed to PEG. D. as the most common compatible solute. 2012). as in PEG. 2008). PEG-treated Impatiens accumulates samples were resolved by NATIVE PAGE and stained for POX activity Pro (Fig. 2008.5%).6%) and counteracted 2008) plants exposed to drought.. and signaling (Verbruggen and Hermans. and H2O2 accumulation (Fig. 3a). because Pro contributes to clearly biphasic effect on POX activity in plants under mild and moder. peroxisomes. and G. Interestingly. Since this result suggested that drought and SA osmoprotection. brane damage. D. 2014)...001) The SOD activity was also elevated under stress. 2012). That PEG et al.1%). and I. control.. The accumulated ROS may of water stress (Farooq et al. Statistically significant differences from control values (LSD test. but unlike CAT it is reduced. 5d). 2. E. ate stress (Fig. Herrera-Vásquez et al.05.

2012. This response was evident in PEG.... 2012). in several of cell expansion. drought induced CAT.. which is com- vate their antioxidative enzymes: SOD. 2002).. 2011). 2013. However... as well as isoform 4.. CAT was the first identified salicylic acid to (Hayat et al. wheat (Marcińska et al... 2012). where CAT activity was very high. for example. it impairs mitosis. Bidabadi et al. Van den Berg and Zeng. which chemical level. width.. Significant reduction of tense stress. et al. peroxidative lipid damage (Fig. Klessig. 2013). SA reduced CAT activity in Celosia argentea plants e. and POX in tomato seedlings (Hayat et al. Odjegba and Adeniyi. since drought or PEG cause an increase in H2O2 content of I.2. ⁎⁎P b 0. water stress was shown for banana. (Piwowarczyk et al. Manohar et al. Our results show that SA alleviates PEG-imposed drought and oxida- Physiological drought imposed by PEG caused a decrease in chloro.001). walleriana shoots were grown on MS media supplemented with increasing PEG and/or SA concentrations for 60 days prior to analyses. Liu et al.. 2008. 2012). 3c and d). 2008). 2013) and banana (Bidabadi et al. hydroponic solution reduced diverse growth parameters in different Marcińska et al. Likewise. 2008.01. 2000. (Fig. Likewise. than in unstressed plants chlorophyll content is commonly recorded in drought-stressed plants. 2012. that was previously shown to appear after virus elimination mechanisms (Milošević et al. Exogenous SA inhibited CAT activity in Impatiens in a is considered as typical symptom of oxidative stress causing pigment dose-dependent manner. tards growth because it causes turgor loss with consequent obstruction SOD. In I. and root length of the plantlets as MDA increase in many species (Hayat et al. 2013). Antonić et al.. in mustard (Alam et al. resolvable by NATIVE PAGE (Fig. Statistically significant differences from control values (LSD test. I.. 2008). 2006. Marcińska In response to elevated ROS (particularly H2O2).. banana (Bidabadi et al. 2013). 2011). Piwowarczyk et al. walleriana (Fig.230 D. Alam et al. but particularly isoforms B and I. Data represent mean ± SE (n = 15). This finding is in concordance with In this study. Drought stress also impairs development. walleriana shoots (Figs. SA ameliorates PEG-induced drought stress through different H. 1 and 2). species (Bajji et al. 3. 2005). as well as photosynthesis (Farooq et al. SA was found to increase CAT activity. 2013). and affects nutrients acquisition by woody species (Liu et al. CAT. Similarly. 2011. 2015). Similarly... 1994.) for the same PEG treatment are marked by asterisks (⁎P b 0. walleriana. Effects of polyethylene glycol (PEG) and salicylic acid (SA) on relative water content (RWC) (a). / South African Journal of Botany 105 (2016) 226–233 Fig. and Lathyrus (Sakthivelu treated Impatiens. where all levels of drought stress increased all three et al... Celosia argentea binding protein (SABP) sensitive to SA inhibition (Sanchez-Casas and (Odjegba and Adeniyi.. 5d). tive stress and that the SA effects are evident from whole-plant to bio- phyll and total pigments content in I. This suggests similar regulation of POX isoforms in Impatiens during oxidative stress caused by biotic and abiotic factors. 2012. leaf water loss (LWL) (b). 2009). toma. PEG reduced height. exposure to PEG induced all 8 POX isoforms accumulation may also negatively affect growth (Maggio et al. (Odjegba and Adeniyi. the addition of PEG to the medium or Bidabadi et al.. and ⁎⁎⁎P b 0. (Burnett et al. and more effectively in plants exposed to in- photo-oxidation and chlorophyll degradation. soybean.. 2014). tolerant cells acti. as well Impatiens. 2012). in . which were also strongly induced by viral infection. 5a).. The reduction of PR under of ascorbate–glutathione cycle.. Lathyrus sp. PEG in the medium retarded growth and development literature data. chlorophyll content of leaves (c). Drought re- studied enzymatic activities (Fig... 5). and total pigments content of leaves (d) of in vitro grown Impatiens. In addition.g. 2011) and in Ctenanthe setosa (Kadioglu roots. Pro et al. studies. 2014). in hydroponically grown in mustard (Alam et al.05. 4b). 2012). in a number of similar and woody species (Liu et al. and POX and the enzymes monly recorded as PR of shoots in vitro.

2011. 2004). Our results also suggest that SA signaling leading to regulation of POX isoforms overrides drought/ROS signaling. the first (oxidative) phase in SA signaling is characterized by a transient increase in ROS. 2012). 2011. due to inhibition of isoforms A. 2008.. CAT. This is important. Hayat et al.01). and by protecting cell membranes against lipid to SA (Chen et al. 2012). 2008). 4a). SA also reduced MDA in drought- stressed mustard (Alam et al. 2004. stress-induced H2O2 accumula- tion promotes SA biosynthesis (Herrera-Vásquez et al. 2008) and wheat (Shakirova and Sakhabutdinova. 2012).g. 5c and d).. as in Impatiens (Fig. but in tomato (Hayat et al. walleriana PEG concentrations on RWC (Fig. and in other cases (Ananieva et al. Kadioglu et al. Miura and Tada. and tomato (Hayat et al. One of prominent SA effects in Impatiens is complete protection against membrane lipid peroxidation. and I (Fig. function as secondary signals to enhance antioxidative activities—SOD. it was shown that SA generally increases total POX. SA (endogenously produced or exogenously supplied) initially has a prooxidant role. 3b).. 2013. 4. resulting in net induction of POX activity in unstressed plants. while in other studies SA generally had a stimulating effect on these enzymes. Differential regulation of POX isoforms in Celosia argentea (Odjegba and Adeniyi. in mustard (Alam et al. 2013). SOD. In the second (antioxidative or reductive) phase. POX. Demiralay et al.. and Celosia argentea (Odjegba and Adeniyi. Hayat et al. The potential of due to induction of isoforms C. Namely. 2015).. and G and net reduction of POX SA to sustain high RWC under physiological drought was even more activity in severely stressed plants.. to- mato (Hayat et al. Hayat et al. since in similar experiments SA was found to slightly induce SOD (Ananieva et al. as compared to untreated plants exposed to the same water stress (Fig. 5b). 3a) but was quite effective in (Fig. Horvath et al. 1997) while. Statistically significant differences from control values (LSD test. In our experimental system.. SA helps maintaining hydration and RWC under stress. banana (Bidabadi et al. exoge- nous SA had no effect on Pro level in unstressed plants.. Herrera-Vásquez et al. 2013). in drought-stressed Ctenanthe setosa leaves (Kadioglu et al. B. SA partially reversed the effects of high SA differently affected different POX isoforms in I... Demiralay et al.. in turn. 2015). which is an atypical result. it should be noted that both PEG and SA treatments of Impatiens lasted for 60 days. 2013).... In Impatiens. 2008). particu- larly H2O2 (Sanchez-Casas and Klessig. and the ascorbate–glutathione cycle enzymes (Ananieva et al. including drought (Herrera-Vásquez et al.. SA decreased SOD activity (Fig. 2013). D. such as enhanced Pro accumulation. 2008. peroxidation. pronounced in some other studies. 4c) and banana (Bidabadi et al. The reduction of MDA is likely a consequence of discussed SA- mediated ROS scavenging. 2009).. Data represent mean ± SE (n = 15). and ⁎⁎Pb0. 2015). 5c and d). walleriana mulation is low to moderate. E. Demiralay et al. particularly when drought-induced Pro accu- level (a). in unstressed barley seedling (Ananieva et al. 5d). Namely. malondialdehyde (MDA) level (b). 2014). resulting in enhanced ROS scavenging (Herrera-Vásquez et al. since total POX activity and POX zymograms of 3 mM SA-treated plants are virtually the same regardless of the PEG treatment and are com- posed of SA-inducible activities only (Fig. To explain why in our experimental system SA reduced total CAT. 2014.. 2011).. as in PEG-treated mustard seedlings (Alam et al. 2004. as it promotes ROS production by stimulating extracel- lular POX and by direct inhibition of two main H2O2 detoxifying enzymes—SABPs catalase and ascorbate peroxidase (Miura and Tada. but other means of membranes protection may also be involved. 2012). SA can also have indi. Alam et al. ex- ogenous SA generally stimulates accumulation of Pro in stressed plants Fig.... 2011.. 2003). 2012). 2007. 4c). 2013). drought-stressed Ctenanthe setosa (Kadioglu et al. wheat (Marcińska et al. Antonić et al. By improving the potential of plant cells for Pro accumulation and 2008. and consequent decline in antioxidative activities (Fig. as discussed later.) for the same PEG treatment are marked by asterisks (⁎P b 0. 2013). Kadioglu et al. 2004. Kadioglu et al. knowing that SA exhibits an ambiv- alent or biphasic action in several stress models. measured as MDA accumulation. 2015)... under all stress levels (Fig. which is a very long period in comparison to treatments in other discussed studies. In Impatiens (Fig. Different CAT isoforms may differ in sensitivity osmotic adjustment... 4b). banana (Bidabadi leaves. Thus. 2012). 5).. H. and certain POX activities.. / South African Journal of Botany 105 (2016) 226–233 231 Impatiens was also demonstrated during viral infection and virus elimi- nation (Milošević et al.05 et al. 2013) and in both unstressed and stressed tomato seedlings (Hayat et al. Effects of polyethylene glycol (PEG) and salicylic acid (SA) on endogenous H2O2 (Misra and Saxena.. 2008). Demiralay SA increased Pro content in unstressed plants as well. This complex interplay between SA and ROS signaling can explain the decrease in H2O2 in Impatiens after prolonged SA treatment.. and proline content (c) in I. SA in- creases reducing power (GSH/GSSG ratio).. et al... In other studies. and tomato (Hayat . maintaining LWL in water-stressed plants (Fig. e. The produced ROS.. 2011. D.. 2013). rect effects on antioxidative activities. 1994.

SA had little or no effect on RWC in un. The label “F” is intentionally omitted so that the isoform labels would correspond to previously published work on I. Marcińska et al. or even (Khan et al.. SA promoted shoot growth and proliferation at 0. that 1. at SA alone did not affect PR. the SA-induced increase of PR was eminent in all index and increased electrolyte leakage. Kang et al. and 3% PEG. and 3 mM SA control (Sakhanokho and Kelley. 2012). drought reduced membrane stability (Fig. 2003. albeit to different de. However. SA had little or no effect stressed plants (Bidabadi et al. seedling grees (Hayat et al. Marcińska et al. Odjegba and Adeniyi. and sampling other effects might be indirect. and peroxidase (POX) (c) activities were assayed spectrophotometrically as described in Section 2. the effect of sion of root cells.. Alam et al. Effects of polyethylene glycol (PEG) and salicylic acid (SA) on activity of antioxidative enzymes in I. and the applied SA concentration but are often tion with multiple receptors or signaling pathways that control promoting (Hayat et al. e. In addition. Data represent on graphs are means ± SE (n = 15). none. Rivas-San Vicente and Plasencia. 2011.. stained with guaiacol and labeled as A–I (d). it retarded inhibitory. ⁎P b 0. 2011). through regulation of hormonal time (Hayat et al. In Hibiscus water loss (Demiralay et al. Kang et al. peroxidase isoforms were separated by NATIVE PAGE. effects (Hayat et al.. 2011)... 2015). drought and salt stress in wheat (Shakirova and Sakhabutdinova. ⁎⁎P b 0. and banana (Bidabadi nificant effect on different growth parameters in corn and soybean et al. 2012). et al.. enhanced mitotic activity and exten- 2012. 2012... 2013).. 2).. 2003). 2010.01. FW. shoot culture. Alam on height.5 mM In Impatiens. 2008). 2011. 2013. Marcińska et al. respectively stressed and salt-stressed maize seedling.. maize (Khodary. growth and development (Rivas-San Vicente and Plasencia. 2013). 3c). 2012). Literature suggests that exogenous SA invariably in. icantly: it may be promotive. the presence of PEG. 3c and d). argentea (Odjegba and Adeniyi. as in Impatiens (Fig. The effect of SA on photosyn. Bidabadi et al. 2012). 2008. Statistically significant differences from control values (LSD test.232 D.. growth. when applied to un- overcompensated damaging effects of 1.. SA was so effective in protecting photosynthetic concentration.. In our experimental system. number of leaves. . while when applied as Na-salicylate. including Impatiens (Fig. It was ited ameliorating effect on these parameters in PEG-stressed plants shown that in tomato seedlings. based on enhanced antioxidative defense and conse. It is likely that some of the SA effects thetic pigments depends not only on the stress conditions and the on growth are a consequence of its water-preserving effects. C. 2013.001) for the same PEG treatment are marked by asterisks. 2. On the contrary. as in mustard and some other species (Rivas-San Vicente growth of Salvia officinalis shoots even at 30 μM concentration and Plasencia. 2009). and lim- et al. while SA overcame these treatments. walleriana leaves from 60-day-old shoot culture. (Khodary. 2003). 3a). 2013).. 5. even when applied in concentration as high as 10 mM creases chlorophyll content in water-stress plants. SA had no sig- 2004). 2003).4. walleriana POX isoforms (Milošević et al. On the other hand. 2. SA enhanced all growth pa- (Fig. In a similar experimental setup with banana shoots culture. as in corn and soybean (Khan et al. 2010. superoxide dismutase (SOD) (b). / South African Journal of Botany 105 (2016) 226–233 Fig.. 2012. However. and FW of the unstressed plants. while studied species but also on the applied SA concentration. 2004). (Kračun-Kolarević et al. the RWC-preserving effect of SA is. Rivas-San Vicente and Plasencia. 2013). but 1 mM SA slightly retarded growth in comparison to pigments from drought-imposed damage.. and DW of seedlings. but significantly enhanced FW. 2012).. Odjegba and Adeniyi. rameters. 2013)..g. Exogenous SA also improved germination. Antonić et al. leves in stressed plants (Shakirova and Sakhabutdinova. Differ- Plant growth responses to exogenous SA vary depending on species. whereas in least partially. improved elements of yield structure. 2012. However.05.. 2008). ent effects of SA on plant growth are probably the result of SA interac- developmental phase. and ameliorated exogenous SA on chlorophyll content in unstressed plants varies signif. Thus.. Total catalase (CAT) (a). Alam et al. and ⁎⁎⁎P b 0. the shoot tips responded positively to SA by signif- quent preservation of membranes integrity and reduced leakage and icant increase of both PR and FW (Bidabadi et al. 2012.

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