ACTA BOT. CROAT. 80 (1), 2021 1 Acta Bot. Croat. 80 (1), 1–11, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-029 ISSN 0365-0588 eISSN 1847-8476 Silver nanoparticles affect germination and photosynthesis in tobacco seedlings Renata Biba1, Mirta Tkalec1, Petra Cvjetko1, Petra Peharec Štefanić1, Sandra Šikić2, Dubravko Pavoković1, Biljana Balen1* 1 University of Zagreb, Department of Biology, Faculty of Science, Horvatovac 102a, 10000 Zagreb, Croatia 2 Dr. Andrija Štampar Institute of Public Health, Department of Ecology, Mirogojska cesta 16, 10000 Zagreb, Croatia Abstract – Extensive commercialization of silver nanoparticles (AgNPs) raises the risk of their accumulation in the soil-plant system. Once released into the environment, AgNPs are prone to chemical transformations, which make it hard to determine whether their phytotoxic effects are purely NP-related or a consequence of released Ag+ ions. In this study the effects of 25, 50, 75, 100 and 150 μM AgNPs and AgNO3 on seed germination and early growth of tobacco (Nicotiana tabacum L.) seedlings were compared. Additionally, the effects on photosynthetic performance and pig- ment content were investigated. Germination rate and index values indicated delayed and slower germination in some AgNP treatments. Lower AgNP concentrations stimulated root growth, but induced a prominent reduction in fresh weight. By contrast, all AgNO3 concentrations inhibited root growth but only the higher ones decreased fresh weight. Obtained results imply that the observed AgNP toxicity could be ascribed to NP form and can be correlated with high AgNP stability in the solid medium. On the other hand, the majority of AgNP and AgNO3 treatments induced an in- crease in chlorophyll content that was accompanied by significantly lower values of relative electron transport rate and coefficient of photochemical quenching, implying an inhibition of the electron transport chain. A similar impact of AgNPs and AgNO3 on photosynthesis can be correlated with lower stability of AgNPs in a liquid medium, resulting in AgNP aggregation and dissolution of Ag+ ions. Keywords: chlorophyll fluorescence, germination, Nicotiana tabacum, photosynthetic pigments, silver ions, silver nanoparticles Introduction The rapid development and extensive commercializa- tion of engineered nanomaterials (ENMs) have expanded their application in industry and daily life, raising serious concerns about their impact on the environment and hu- man health (Scheringer 2008, Wiesner et al. 2009). Among the various types of ENMs, silver nanoparticles (AgNPs) are involved in nearly 25% of consumer products (Vance et al. 2015). Special physicochemical properties of Ag nanomate- rials play a crucial role in their antibacterial activity, which is being utilized in the environmental, biomedical and in- dustrial sectors (Deshmukh et al. 2019). Increased use of AgNPs raises the risk of their discharge into the environ- ment and accumulation in the soil-plant system (Yang et al. 2017, Lv et al. 2019). Through plants AgNPs could be trans- ported and accumulated in high trophic-level consumers, including humans (Rico et al. 2011, McKee and Filser 2016). Previous studies have shown both positive and negative impacts of AgNPs on plant metabolisms, mainly depend- ing on their concentration, size, shape and coating (Pallavi et al. 2016, Jasim et al. 2017, Cvjetko et al. 2017, 2018, Pe- harec Štefanić et al. 2019). Experimental methodology (growth medium, exposure method, exposure time) as well as plant system used (species and developmental stage) al- so significantly affect AgNP phytotoxicity (Yan and Chen 2019). AgNP suspensions are prone to chemical transfor- mations (oxidation, dissolution, aggregation and agglom- eration) (Levard et al. 2012, Gorham et al. 2014) making it * Corresponding author e-mail: bbalen@biol.pmf.hr BIBA R., TKALEC M., CVJETKO P., PEHAREC ŠTEFANIĆ P., ŠIKIĆ S., PAVOKOVIĆ D., BALEN B. 2 ACTA BOT. CROAT. 80 (1), 2021 dissolved in ultrapure water (ion-free Milli-Q water, 18.2 MΩ.cm resistivity, Merck Millipore, USA) and used as a 100 mM stock solution. Prior to treatments, the concentration of Ag in the AgNP and AgNO3 stock solution was deter- mined by ELAN DRC-e inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer, USA). For prepara- tion of treatment solutions of AgNPs, the concentration of silver was considered in calculations. Plant material and culture treatments In this study we used solidified half strength Murashige and Skoog (MS) nutrient medium (0.2% (w/v) Phytagel, 1.5% (w/v) sucrose; both Sigma Aldrich, USA) (Murashige and Skoog 1962) to study the effects of AgNPs and AgNO3 on the germination and early growth of tobacco (Nicotiana tabacum L. cv. Burley). Nutrient medium was sterilised and supplemented with AgNP or AgNO3 stock solutions to ob- tain 25, 50, 75, 100 and 150 µM concentrations and subse- quently poured into Petri dishes (90 mm diameter) and left to solidify. Tobacco seeds were surface sterilised for 15 min using 50% (v/v) NaOCl (Kemika, Croatia), and then rinsed three times with sterile deionised H2O before being placed on the half strength MS medium. Control seeds were germi- nated on a medium devoid of AgNPs and AgNO3. Two days before the beginning of the experiment, Petri dishes with sown seeds were placed in cold stratification (+4 °C) to pro- mote and synchronise seed germination (Kucera et al. 2005). Germination was monitored for 5 consecutive days, starting from the 3rd day after seed stratification. Seedlings were harvested after three weeks and used for measure- ments of root length as well as fresh and dry weight. All ex- periments were conducted two times. In each experiment, 100 seeds were sown for every treatment with either AgNPs or AgNO3 (200 seeds in total per treatment). For analysis of effects of AgNPs and AgNO3 on photo- synthesis and photosynthetic pigments and for measure- ment of Ag content, sterilized and stratified seeds were placed in sterile 100 mL Erlenmeyer flasks filled with 5 mL of liquid half strength MS medium and left to germinate on a shaker. Germinated seeds were grown for three weeks in the same Erlenmeyer flask, which was periodically supple- mented with fresh sterile nutrient medium. After three weeks, the nutrient medium was replaced with fresh sterile liquid half strength MS medium supplemented with either AgNPs or AgNO3 in the above-mentioned concentrations. Seedlings were treated for 7 days. During the experiments, all plant material was kept in the culture room at 24 ± 1 °C with 16/8 h light/dark cycles and 90 µmol m–2 s–1 light intensity. AgNP stability in solid and liquid culture medium AgNPs stability in the solid nutrient medium was ana- lysed as previously reported (Peharec Štefanić et al. 2018). Briefly, one mL of half strength MS medium, solidified with Phytagel (agar substitute) and supplemented with AgNP stock solution to obtain a 150 µM concentration (the high- harder to determine whether the effects of AgNPs are pure- ly nanoparticle-related or a consequence of silver ion re- lease from the nanoparticles. So far, research has provided evidence for both Ag+ ion- and AgNP-specific toxicity (Tkalec et al. 2019). Ag+ ions released from AgNPs can in- duce oxidative stress through excessive reactive oxygen species (ROS) production (Park et al. 2009), disturb cell function by binding to cell components and modifying their activities (Montes et al. 2017) and affect photosynthe- sis through competitive substitution of Cu+ ions in plasto- cyanin (Sujak 2005, Jansson and Hansson 2008). However, in some cases AgNPs proved to be more toxic than Ag+ ions at the same concentrations, due to the inhibition of apo- plastic trafficking caused by the clogging of pores and bar- riers in the cell wall or plasmodesmata (Tripathi et al. 2017, Ruotolo et al. 2018). High sensitivity to AgNPs caused by inhibition of pho- tosynthetic processes was shown in several algae and plant species (Navarro et al. 2008, Oukarroum et al. 2012, Jiang et al. 2017, Dewez et al. 2018). Reduced photosynthetic ac- tivity and decreased ATP and NADPH synthesis can dis- turb the biochemical and physiological processes needed for cell growth, which in the end affects plant development (Gerst et al. 1994, Stirbet and Govindjee 2012). Measure- ment of chlorophyll a fluorescence together with the content of photosynthetic pigments can provide valuable insight in- to the mechanism of AgNP phytotoxicity, and coupled with germination percentage, root elongation and mass measure- ments, fast and reliable toxicity indicators, gives a more comprehensive view of the effects of AgNPs on plants (Wang et al. 2001, Dewez et al. 2018). To elucidate the nature of AgNP-phytotoxicity, the ef- fects of citrate-coated AgNPs and ionic Ag in the form of silver nitrate (AgNO3), both applied in concentrations of 25, 50, 75, 100 and 150 μM, on seed germination, early growth as well as on photosystem II (PSII) performance and the photosynthetic pigment content of tobacco (Nicotiana tabacum L.) seedlings, were compared. The significance of the work we are reporting on derives from interactions that have received little attention to date; the difference of pos- sible phytotoxic effects of silver nanoparticles and ionic sil- ver on germination and early growth as well as on photo- synthesis. As object of our study we chose tobacco, not only an economically interesting and important plant but also a frequently used model organism in abiotic stress re- search (Gichner et al. 2004, Peharec Štefanić et al. 2012, Tkalec et al. 2014). Materials and methods AgNPs and AgNO3 suspensions All experiments were performed with commercial Ag- NPs with citrate coating (50 nm Citrate BioPure Silver Nanospheres, Nanocomposix, San Diego, CA, zeta potential of –47.8 mV). The concentration of AgNP stock solution was 9.27 mM. AgNO3 (Sigma Aldrich St. Lois, MO, USA) was AgNPs AFFECT TOBACCO GERMINATION AND PHOTOSYNTHESIS ACTA BOT. CROAT. 80 (1), 2021 3 est applied AgNP concentration in this study), was prepared in a 1 cm quartz cuvette for spectrophotometric absorbance measurements. The cuvette was sealed with Parafilm M to prevent medium from drying and was kept in the same con- ditions as plant material during 5 days. Measurements of spectrophotometric absorbance were performed using the UV-visible spectrophotometer (ATI Unicam, Cambridge, UK) in the wavelength range of 300–800 nm. For instru- ment zeroing, solid half strength MS medium devoid of Ag- NPs was applied. Stability of AgNPs was monitored regu- larly during the period of 5 days. For measurements of AgNP stability in the liquid half strength MS medium a similar procedure for spectropho- tometric measurements was applied. The differences were that a liquid half strength MS medium was used instead of a solid and that the cuvette was kept for 7 days in the same conditions as the plant material. Measurements of spectro- photometric absorbance were performed in the abovemen- tioned wavelength range. For instrument zeroing, liquid half strength MS medium devoid of AgNPs was applied. Stability of AgNPs was monitored regularly during the pe- riod of 7 days. To confirm the data obtained by spectrophotometric analysis, the size and charge of nanoparticles in 150 µM AgNP solution in liquid half strength MS medium were measured with the dynamic light scattering (DLS) tech- nique using a Zetasizer Nano ZS (Malvern, UK) equipped with green laser (532 nm). Intensity of scattered light was detected at the angle of 173°. All measurements were con- ducted at 25 °C. The data processing was done with the use of the Zetasizer software 6.32 (Malvern instruments). Mea- surements were performed at 0 and 15 min as well as 1, 5 and 24 h after the solution of AgNPs in nutrient medium was prepared. Results are reported as an average value of 10 measurements and the size distributions are reported as vol- ume distributions. The charge of AgNPs was evaluated by measuring electrophoretic mobility of AgNPs and results are reported as an average value of 5 measurements. In addition, AgNPs were visualized in nutrient media of all tested AgNP concentrations after exposure of tobacco seedlings in a liquid medium using a FEI Morgagni 268D electron microscope. TEM samples were prepared by depos- iting a drop of the sample suspension on a Formvar®/Carbon copper grid. Samples were air-dried at room temperature. Germination parameters Seed germination was monitored for 5 days, each day at the same time. Seeds were considered germinated when the radicle emerged from the seed. Germination percentage was calculated using the formula: Germination percentage (%) = final number of germi- nated seeds/total number of seeds × 100. To calculate germination index (GI) and germination rate (T50), daily counts of germinated seeds were used by employing the following formulas (Farooq et al. 2005): GI = ∑ Nt (number of germinated seeds on the t day)/Dt (germination days) T50 = ti+{[(N/2)-ni]*(ti-tj)/ni-nj} where N is the final number of germinated seeds, ni and nj are cumulative number of seeds germinated by consecutive counts at times ti and tj, considering that ni