Impaginato 563 Adv. Hort. Sci., 2018 32(4): 563-568 DOI: 10.13128/ahs-22468 A preliminary investigation on developmental and biochemical responses of Amsonia orientalis to ultraviolet-C irradiation A. Acemi 1 (*), Y. Avcı Duman 2, Y. Yüzügüllü Karakuş 1, F. Özen 1 1 Department of Biology, Faculty of Sciences and Arts, Kocaeli University, 41380 Kocaeli, Turkey. 2 Department of Chemistry, Faculty of Sciences and Arts, Kocaeli University, 41380 Kocaeli, Turkey. Key words: antioxidant enzymes, European Bluestar, plant development, tissue culture, UV-C irradiation. Abstract: The present study aims to investigate the developmental and bio- chemical responses of ornamental Amsonia orientalis irradiated with ultravio- let-C (UV-C). Nodal explants of the species were exposed to UV-C irradiation on the first, 15th and the last days of the in vitro culture for 15 (3.47 kJ m-2), 30 (6.94 kJ m-2) and 60 min (13.87 kJ m-2). In general, root lengths and numbers were negatively influenced by prolonged UV-C exposure. However, mean shoot numbers and lengths were slightly enhanced after 15 and 30 min of irradiation. High hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels were observed due to the UV-C exposure. Activities of the antioxidant enzymes, POD (peroxidase) and CAT (catalase) were found to be enhanced whereas SOD (superoxide dismutase) was reduced. These results indicated that UV-C irradia- tion for shorter durations may be carefully used to improve in vitro shoot pro- liferation in A. orientalis. However, it should be noted that longer irradiation durations can trigger stress responses and lipid peroxidation-dependent cell membrane damage which will further result in the plant loss. 1. Introduction Ultraviolet (UV) irradiation is present in sunlight in three different wavelengths which are classified as UV-A (400-315 nm), UV-B (315-280 nm) and UV-C (280-100 nm). UV irradiation may affect growth and meta- bolic processes in plants due to its high quantum energy (Kobashigawa et al., 2011). Since wavelengths below 280 nm are absorbed by the ozone layer they do not reach to the surface of the Earth (Alexieva et al., 2001). However, reduction of stratospheric ozone and, a decrease in the ozone layer may lead to increase in UV-C irradiation reaching the biosphere. (*) Corresponding author: arda.acemi@kocaeli.edu.tr Citation: ACEMI A., AVCI DUMAN Y., Yüzügüllü KArAKUş Y., ÖzEN F., 2018 - A preliminary investigation on developmental and biochemical responses of Amsonia orientalis to ultraviolet-C irradiation. - Adv. Hort. Sci., 32(4): 563-568 Copyright: © 2018 Acemi A., Avci Duman Y., Yüzügüllü Karakuş Y., Özen F. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. received for publication 7 January 2018 Accepted for publication 30 May 2018 AHS Advances in Horticultural Science Short note Adv. Hort. Sci., 2018 32(4): 563-568 564 Beside its germicidal activity, application of UV-C irra- diation at 254 nm wavelength is used for several pur- poses including regulation of fruit ripening in tomato (Tiecher et al., 2013), fruit quality stabilization during storage of fresh-cut watermelon (Artés-Hernández et al., 2010) and retardation of fruit decay in strawberry (Erkan et al., 2008). Its role in growth regulation and modulation of flowering time in ornamental plants was declared since UV-C light application increased branching and the number of flowers in Salvia splen- dens and Viola tricolor (Bridgen, 2016). Also, the antioxidative function was found to be enhanced a f t e r U V - C a p p l i c a t i o n i n S p i n a c i a o l e r a c e a (Kobashigawa et al., 2011). However, the application of UV irradiation can be hazardous in higher doses. Therefore, to better understand the developmental and metabolic responses of horticultural plants to UV-C irradiation, UV-dose-dependent studies in con- trolled environmental factors should be also conduct- ed on other plant species. Amsonia orientalis Decne. [syn. Rhazya orientalis (Decne.) A. DC.] which is also known as European Blue star or Eastern rhazya is an ornamental plant with medicinal properties. It is cultivated more com- monly by gardeners in the USA than those in Europe since it is deer-resistant due to its slightly toxic latex content (Acemi et al., 2016). However, the natural populations of the species were taken under conser- vation by the European Council in the frame of Bern Convention since they are quite limited in nature (Acemi et al., 2017). In the current study, the effects of dose-dependent UV-C irradiation applications on in vitro development and antioxidative enzymes of A. orientalis were investigated to shed light the usability of this treatment in horticultural plant propagation. 2. Materials and Methods Plant material preparation and in vitro UV-C treat- ments Nodal explants were excised from mature individ- uals of Amsonia orientalis growing in the garden of Kocaeli University. The shoots were multiplied by fol- lowing the protocol described by Acemi et al., (2013 a). Nodal explants excised from in vitro-raised shoots were inoculated into the MS (Murashige and Skoog’s) medium (1962) supplemented with 30 g l-1 sucrose and 7 g l-1 of plant agar. The pH of the medium was set to 5.7, and the cultures were incubated for 30 days under the same conditions defined by Acemi et al., (2013 a). The applied UV-C doses were deter- mined from previous reports (lópez-rubira et al., 2005; Artés-Hernández et al., 2010; Bridgen 2014; Castronuovo et al., 2014; Bridgen, 2016). The cul- tures were subjected to UV-C irradiation at the first, 15th and the last days of the culture period. The irra- diation was applied from 30 cm distance for 15, 30 and 60 min per application day by using Sylvania g15W UV-C lamp (λmax 253.7 nm). UV-C treatments were applied from the top of the culture vessels in a sterile biosafety cabinet, and lids of the culture ves- sels were kept opened during treatments to ensure penetration of the irradiation energy. The total accu- mulated irradiation levels were estimated as 3.47, 6.94 and 13.87 kJ m-2 for 15, 30 and 60 min of UV-C irradiation, respectively (Kobashigawa et al., 2011; Severo et al., 2015). Biochemical assays The optimized method of Acemi et al. (2017) was followed to determine malondialdehyde (MDA) as lipid peroxidation product, and H2O2 contents. The crude extract for antioxidant enzyme activities’ determination was prepared by homogenizing the tissue samples (shoots and roots together) in extrac- tion buffer of 50 mM sodium phosphate (pH 7.0) con- taining 0.1 mM ethylenediaminetetraacetic acid (EDTA). After centrifugation of the homogenate at 14,000 g for 15 min at 4°C, the resulting supernatants were collected and used for catalase (CAT), peroxi- dase (POD) and superoxide dismutase (SOD) activity assays. CAT activity was determined according to the method of Aebi (1974). POD activity was assayed using the pyrogallol oxidation method (Kar and Mishra, 1976) while SOD activity was determined by following the method of Dhindsa et al. (1981). Data collection and statistical analysis The morphometric evaluation was done using 30 explants in each repeat. All assays were repeated t h r i c e a n d m e a n v a l u e s w e r e c o m p a r e d u s i n g Duncan’s multiple range test at p<0.05 significance level. The enzyme assays and extract preparation were done on the same day to minimize the loss of enzymatic activities. 3. Results Plant growth and organ development At the end of the incubation period, the mean shoot length slightly increased in response to 15 and Acemi et al. - UV-C irradiation effects on Amsonia orientalis 565 30 min UV-C exposure while this parameter was neg- atively affected by 60 min of exposure. However, increments in the mean shoot length were not statis- tically different than the control group (Fig. 1A). The mean shoot numbers were influenced positively by 15 and 30 min UV-C exposure whereas only the result of 15 min exposure was found to be statistical- ly different than the control group (Fig. 1B). The mean root lengths were found to be decreased due to UV-C application even at the shortest duration. This reduction in root lengths was between 62.5% (15 min) and 95% (60 min) compared to the control (Fig. 1A). In contrast to mean shoot numbers, the mean root numbers reduced gradually in response to the elevated UV-C exposure (Fig. 1B). Lipid peroxidation and H2O2 content Application of UV-C irradiation significantly changed the lipid peroxidation level of the plant. Fifteen and 30 min of UV-C exposures triggered MDA a c c u m u l a t i o n s w i t h o u t s t a t i s t i c a l d i f f e r e n c e s between them. The highest accumulation level was observed from the cultures exposed to UV-C for 60 min (Fig. 2A). H2O2 content increased following UV-C exposure maintaining similar levels within treat- ments (Fig. 2B). Antioxidant enzyme activities UV-C irradiation caused a decrease in SOD activity without statistical differences between treatments (Fig. 3A). POD activity significantly increased due to U V - C e x p o s u r e s . T h e h i g h e s t P O D a c t i v i t y w a s observed in cultures exposed to 15 min of UV-C fol- lowed by a significant decrease at 30 and 60 min of UV-C exposures, which were not significantly differ- ent (Fig. 3B). CAT activity exhibited a similar trend with POD activity. The shortest UV-C exposure caused the highest increase in the CAT activity while longer exposures induced statistically same results (Fig. 3C). Fig. 1 - Effects of UV-C irradiation on Amsonia orientalis growth parameters. A= length of shoot and root, B= Number of shoot and root. The values are represented as means ± SD, different letters denote significant differences (p<0.05). Fig. 2 - Oxidative effects of UV-C irradiation on Amsonia orienta- lis. A= MDA and B= H2O2 contents. The values are repre- sented as means ± SD, different letters denote significant differences (p<0.05). Adv. Hort. Sci., 2018 32(4): 563-568 566 4. Discussion and Conclusions UV irradiation can damage DNA and affect plant growth through various regulatory and/or stress- mediated processes. As one of these processes, cell cycle modulation sourced by DNA damage may par- ticularly slow down the progress from g1 to S phase (Jiang et al., 2011). Therefore, in our results the decrease in the mean shoot length after 60 min UV-C irradiations might be due to the UV-induced DNA damage and impaired cell cycle progress. Differently, mean shoot number was found to be increased in A. orientalis after 15 min of UV-C exposure, which was in accordance with the previous report that found an increment of axillary branching in Viola tricolor (Bridgen, 2016). Our results also showed adverse effects of UV-C on the root growth of A. orientalis. This negative effect can be explained by oxidative degradation and/or inhibited-synthesis of indole acetic acid (IAA); a phytohormone responsible for root proliferation (Berli et al., 2013). This possible inhibition of IAA might also be a result of photo-oxi- dation of the plant growth regulator (Ciurli et al., 2017). The oxidative degradation of IAA starts with a peroxidase-involved decarboxylation process on the side chain or oxidation of the indole ring (Normanly, 2010). In the enzyme assays, elevated peroxidase activity against UV irradiation supports this discus- sion which is still in need of further experiments to be clearly proven. MDA content indicates reactive oxygen species (rOS)-mediated cellular damage considering damage to membrane lipids of stress-exposed plants. The observed increase in H2O2 levels coincided with enhanced MDA levels in A. orientalis. Also, POD and CAT activities were found to be increased to remove r O S t o l i m i t M D A p r o d u c t i o n i n A . o r i e n t a l i s . Degradation of the enzyme proteins and nucleic acids can be started after rOS-induced peroxidation of the cell membrane lipids. At the same time, H2O2 could activate mitogen-activated protein kinases (MAPKs) in plants, leading to an enhanced antioxi- dant defense system (Nie et al., 2013). However, excess accumulation of H2O2 causes cellular damage (gong et al., 2001). The increase in both H2O2 and MDA levels indicates cellular damage in A. orientalis. Excessive rOS in UV-C-exposed plants may be pro- duced because of disruption in metabolic activities or increased activity of membrane-localized NADPH-oxi- dase (Kalbina and Strid, 2006). The increment in the activities of CAT and POD enzymes did not seem to limit rOS production in A. orientalis since H2O2 accu- mulation was consistently found at high levels. As a defense mechanism against environmental stress factors, plants favor the production of antioxi- dant enzymes (Berli et al., 2013). Maintenance of the Fig. 3 - Effects of UV-C irradiation on enzymatic antioxidant acti- vities in Amsonia orientalis. A= SOD, B= POD and C= CAT activities. The values are represented as means ± SD, dif- ferent letters denote significant differences (p<0.05). Acemi et al. - UV-C irradiation effects on Amsonia orientalis 567 antioxidant defense system to cope with rOS plays a significant role in keeping the cell membranes stabi- lized. The antioxidant enzymes SOD, CAT, and POD a r e w i d e l y d i s t r i b u t e d i n a l l h i g h e r p l a n t s a n d involved in decomposition of different forms of rOS (Foyer and Noctor, 2000). Excessive production of O2– triggers SOD enzyme activity which converts superox- ide radicals into either O2 or H2O2 while the excess accumulation of H2O2 is prevented by catalase and/or the ascorbate-glutathione cycle enzymes (Ma et al., 2014). Although the O2− content was not determined in our study, the inhibition of SOD activity by UV irra- diation might be due to increased O2− content. POD is involved in such processes like lignification and toler- ance to environmental stresses in higher plants. During UV exposure, elevated POD and CAT activities showed that activities of both enzymes can be trig- gered in A. orientalis. The bulk H2O2 removal activity of CAT in the cell is followed by the scavenging action of POD on the H 2O 2 which is not taken by CAT (Willekens et al., 1997). In this report, this coopera- tion between both enzymes is also shown in A. orien- talis. Thanks to in vitro plant tissue culture technique, several conservation studies on A. orientalis were conducted and a high number of individuals were propagated thereby populations in Turkey were con- served (Acemi et al., 2013 a, b). The present study has revealed that although short-term application of UV-C irradiation can enhance shoot induction, it lim- its root growth and triggers oxidative stress at extended exposure durations. Therefore, in further detailed studies on A. orientalis UV-C radiation should be applied for less than 30 min (6.94 kJ m-2). 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