The oxidative stress in . The participation of prenyllipid antioxidants in the response to juglone. Beatrycze Nowicka, Bartosz Pluciński, Paulina Kuczyńska, Jerzy Kruk Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland, corresponding author e-mail: [email protected]

Introduction t=0 h t=0 h 1.0 3.0 t=2.5 h t=2.5 h · Allelopathy, according to narrow definition, is the t=5 h t=5 h 2.5 t=7.5 h 0.8 t=7.5 h ] effect of one plant species on another through the release l ] m 2.0 l /

Results of g 0.6 m / of chemical compounds into the environment [1]. Broader m [ g m b

1.5 [ r definitions cover also interactions between plant, microbes experiment 1 + a 0.4 a C l 1.0 and fungi [2]. Allelochemicals are secondary metabolites, h C 0.2 often produced as inactive precursors and released 0.5 thorough leaching from aerial parts, root exudation, juglone 0.0 0.0 volatile emissions and decomposition of residues. The control juglone control juglone modes of action of certain allelochemicals are various, one Fig. 1. The effect of exposure to juglone on the content of Chl a + b (left) and total carotenoids (right) during 7.5 h of incubation in weak light. of them is the induction of oxidative stress [3]. The data are means ± SD (n=3). Car, total carotenoids; Chl a + b, sum of chlorophyll a and b; cont, control series. 0.12 0.0014 0.10 t=0 h t=0 h t=0 h t=2.5 h t=2.5 h t=2.5 h · Oxidative stress is a situation, when there is a serious 0.10 0.0012 t=5 h t=5 h 0.08 t=5 h ] ] l l t=7.5 h t=7.5 h ] t=7.5 h imbalance between the reactive oxygen species (ROS) 0.0010 l m m

/ 0.08 / m / s s l l s

l 0.06

production and detoxification, resulting in excessive o

o 0.0008 o m -- 0.06 m m n n [ [ concentration of ROS in cells [4]. ROS are both radical (O , n 2 0.0006 [ c t c 0.04 o t

1 o o

T 0.04 T

Q

HO , OH) and non-radical ( O , H O ) forms, usually - 2 2 2 2 - 0.0004 g P a 0.02 generated as by-products of aerobic metabolism. They can 0.02 0.0002 play a beneficial role in signalling and pathogen defence, 0.00 0.0000 0.00 but can also damage cellular components. The main site of control juglone control juglone control juglone ROS production in plant and algal cells are chloroplasts; Fig. 2. The effect of exposure to juglone on the content of a-tocopherol (left), g-tocopherol (middle) and total plastoquinone (sum of plastoquinone and plastoquinol, right) during 7.5 h of incubation in weak light. The data are means ± SD (n=3). cont, control series, PQ , mitochondria and peroxisomes being other important tot sources of ROS [5]. total plastoquinone; a-Toc, a-tocopherol; g-Toc, g-tocopherol. 200 t=0 h

p t=2.5 h

· Prenyllipid antioxidants belonging to isoprenoid h

L t=5 h

- 150 The exposure to juglone led to significant decrease in and chromanols are amphipathic compounds, Y t=7.5 h P

S photosynthetic pigments and prenyllipid content, occurring ubiquitously in membranes. Prenylquinones act f o 100 e as mobile electron and proton carriers and c which was accompanied with the increase in LOOH n e c cofactors. Members of both groups also participate in signal s content, which points at proceeding lipid e

r 50 o

transduction. These compounds are potent antioxidants, u l peroxidation. 1 F able to quench and scavenge O2 and scavenge oxygen and 0 lipid radicals. Important prenyllipid antioxidants present in control juglone chloroplasts are tocopherols (a-Toc and g-Toc) and Fig 3. The effect of exposure to juglone on Spy-LHP fluorescence. The fluorescence was normalized on the signal plastoquinone (PQ)/plastoquinol (PQH2) [6] obtained for t=0 samples. The data are means ± SD (n=3). · Juglone, 5-hydroxy-1,4-naphthoquinone, belong to the most recognized allelochemicals. Its inactive precursor is control D control D control HL control HL produced by trees of genus and released into the 120 juglone D 120 juglone D juglone HL juglone HL t

environment, where hydrolysis and oxidation results in n

e 100 100 t t n n e o t c formation of active compound. Juglone was shown to n o 1

Results of t 80 c 80 n 1 t o n inhibit germination and growth of sensitive plants, inhibit d o e z d i 60 60 l

experiment 2 e a z i l respiration and photosynthesis and cause wilting [7]. The m r a o m n 40 r 40 o b effect of juglone is pleiotropic and mechanisms of its n + r a a l C

h 20 20 have not been fully elucidated yet [8]. Juglone is a C strong redox cycler, which means that in vivo it can be 0 0 t1 10 min 20 min 30 min 40 min t1 10 min 20 min 30 min 40 min easily reduced to semiquinol, which can further reduce Fig. 4. The effect of simultaneous exposure to juglone and high light on the content of Chl a + b (left) and total carotenoids (right) during 40 O2,to [3,9]. Reduced juglone can also react with min of incubation in high light or in darkness. The pigment content is expressed as a percentage of content after 1 h of preincubation. The glutathione leading to depletion of this antioxidant [2]. The data are means ± SD (n=3). Car, total carotenoids; Chl a + b, sum of chlorophyll a and b; cont D, control series kept in darkness, cont HL, occurrence of oxidative stress in juglone-exposed plants control series kept in high light, juglone D, series with juglone kept in darkness, juglone HL, series in high light. has been observed [10-13]. Other mechanisms of juglone control D control D control D control HL control HL 140 control HL juglone D juglone D toxicity are: inhibition of p-hydroxyphenylpyruvate 120 120 juglone D juglone HL juglone HL 120 juglone HL t

dioxygenase, a key enzyme of PQ and Toc biosynthesis t 100 100 t n n n e e e t 100 t

+ t n n n o o o

[14], inhibition of H -ATPase necessary for keeping of water c c 80 80 c 1 1 1 t t t 80 n n n o o homeostasis [15], inhibition of peptidyl-proly cis/trans o d d d

e 60 60 e e z z z i 60 i i l l l a a isomerases and RNA polymerases [16] a m m m r r r o 40 o 40 o n

n 40 n t c c o t o o T Q T - - P g · Chlamydomonas reinhardtii is a common freshwater a 20 20 20 green microalga, thought of as a model unicellular 0 0 0 t1 10 min 20 min 30 min 40 min t1 10 min 20 min 30 min 40 min t1 10 min 20 min 30 min 40 min photosynthetic organism and often used in environmental Fig. 5.The effect of simultaneous exposure to juglone and high light on the content of a-tocopherol (left), g-tocopherol (middle) and total studies, as it is easy to grow, metabolically profiled and its plastoquinone (sum of plastoquinone and plastoquinol, right) during 40 min of incubation in high light or in darkness. The prenyllipid genome has been sequenced [17]. Our preliminary content is expressed as a percentage of content after 1 h of preincubation. The data are means ± SD (n=3). cont D, control series kept in experiments have shown that C. reinhardtii is sensitive to darkness, cont HL, control series kept in high light, juglone D, series with juglone kept in darkness, juglone HL, series in high light. PQtot, juglone. Therefore, it can be used as a simplified model for total plastoquinone; a-Toc, a-tocopherol; g-Toc, g-tocopherol. observation of some modes of juglone action. 8000 control D control HL 7000 The simultaneous application of high light and juglone juglone D

t juglone HL

· The aim of the study was to examine the impact of n 6000

e led to a significant decrease in total carotenoid, total t n o juglone exposure on photosynthetic pigments, prenyllipid c 5000 1 t PQ and Toc content. High light alone did not cause a n content and lipid peroxidation in C. reinhardtii o 4000 d e z i

l decrease in carotenoid and prenyllipids, while in (experiment 1) and to examine the combined effect of a 3000 m r o juglone and high light stress (experiment 2). n 2000 series with juglone alone only a slight decrease (or no Q T - a 1000 decrease) was observed. What is more, there was a

0 t1 10 min 20 min 30 min 40 min significant accumulation of an oxidation product of a- Materials and methods Fig 6.The effect of simultaneous exposure to juglone Toc, a-tocopheryloquinone (a-TQ) in series with A C. reinhardtii strain 11-32b (SAG collection, Goettingen, Germany) was and high light on the content of a-tocopherolquinone, an juglone + high light, while there were no a-TQ grown in a modified Sager-Granick medium with 5 mM HEPES pH 6.8 and oxidation product of a-tocopherol, during 40 min of 0.2 mM sodium acetate as described in [18]. The photosynthetic incubation in high light or darkness. The prenyllipid increase in darkness (both in juglone and control pigments were extracted with acetone [19] and their concentration was content is expressed as a percentage of content after 1 h series) and much smaller increase in high light determined spectrophotometrically, as described in [20]. Before the of preincubation. The data are means ± SD (n=3). a-TQ, control. experiments, cultures were diluted with a fresh medium to get a final a -tocopherolquinone. chlorophyll concentration of 2.5 mg Chl/ml. 10 mM juglone in DMSO was prepared just before application. Juglone was added to the algal culture to obtain final concentration 20 mM. Conclusions In experiment 1 juglone-treated and control cultures were put on the References: 2 shaker and kept under the weak light (35 mmols photons m /s). Samples [1] Rice EL (1979) Allelopathy: an update, Bot Rev 45:15–109. ·Exposure to juglone causes oxidative stress in C. [2] Macías FA, Molinillo JM, Varela RM, Galindo JC (2007) Allelopathy - a natural alternative for weed control, Pest Manag Sci 63:327-348. were taken a t=0, 2.5, 5, 7.5 h. [3] Weir TL, Park SW, Vivanco JM (2004) Biochemical and physiological mechanisms mediated by allelochemicals, Curr Op Plant Biol 7:472-479. In experiment 2 juglone-treated and control cultures (in flat plastic [4] Ahmad P, Sarwat M, Sharma S (2008) Reactive oxygen species, antioxidants and signaling in plants, J Plant Biol 51:167- reinhardtii and prenyllipid antioxidants participate in 173. [5] Gechev TS, van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant bottles) were preincubated in the weak light on a shaker for 1 h, then stress responses and programmed cell death, BioEssays 28:1091–1101. [6] Kruk J, Szymańska R, Nowicka B, Dłużewska J (2016) Function of isoprenoid quinones and chromanols during oxidative either kept in darkness (D) or exposed to white high light (HL, 1000 mmols ROS scavenging. stress in plants, New Biotechn http://dx.doi.org/10.1016/j.nbt.2016.02.010. [7] Willis RJ (2000) Juglans spp., juglone and allelopathy, Allelopathy J, 7:1-55. 2 [8] Dayan FE, Duke SO (2009) Biological activity of allelochemicals. In Plant-derived Natural Products, Springer US. pp. 361- photons m /s) for 40 min (on magnetic stirrers). Samples were taken at 384. [9] Koster AS (1991) Bioreductive activation of quinones: a mixed blessing. Pharmaceutisch Weekblad 13:123-126. t=0, after 1 h of preincubation and after 10, 20, 30, 40 min (for HL series) ·High light significantly increases juglone toxicity. This is [10] Babula P, Adam V, Kizek R, Sladký Z, Havel L (2009) Naphthoquinones as allelochemical triggers of programmed cell death, Environ Exp Bot 65:330-337. [11] Chen SY, Chi WC, Trinh NN, Cheng KT, Chen YA, Lin TC, et al. (2015) Alleviation of allelochemical juglone-induced or 20, 40 min (D series). phytotoxicity in tobacco plants by proline, J Plant Inter 10:167-172. probably a result of more effective juglone reduction. [12] El Hadrami A, Kone D, Lepoivre P (2005) Effect of juglone on active oxygen species and antioxidant in susceptible Photosynthetic pigments were determined as described above. Prenyllipid and partially resistant banana cultivars to black leaf streak disease, Eur J Plant Pathol 113:241-254. [13] Mylona PV, Polidoros AN, Scandalios JG (2007) Antioxidant gene responses to ROS-generating xenobiotics in developing and germinated scutella of , J Exp Bot 58:1301-1312. antioxidants were extracted with acetone and analyzed using RP-HPLC, as However, photocatalytic reactions of juglone itself were [14] Meazza G, Scheffler BE, Tellez MR, Rimando AM, Romagni JG, Duke SO, et al. (2002) The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase. Phytochemistry 60:281-288. described in [18,19,21]. For lipid hydroperoxides (LOOH) determination [15] Hejl AM, Koster KL (2004) Juglone disrupts root plasma membrane H+-ATPase activity and impairs water uptake, root respiration, and growth in (Glycine max) and corn (Zea mays), J Chem Ecol, 30:453-471. [16] Chao SH, Greenleaf AL, Price DH (2001) Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks samples were extracted with ethanol, then a method with Spy-LHP also postulated in the literature [7]. transcription, Nucl Acid Res 29:767-773. [17] Hanikenne M (2003) Chlamydomonas reinhardtii as a eukaryotic photosynthetic fluorescent probe was used, as described in [18]. model for studies of heavy metal homeostasis and tolerance, New Phytol 159:331–340. [18] Nowicka B, Pluciński B, Kuczyńska P, Kruk J (2016) Prenyllipid antioxidants participate in response to acute stress ·The decrease of prenyllipid antioxidants may result not induced by heavy metals in green microalga Chlamydomonas reinhardtii, Environ Exp Bot 123:98–107. [19] Nowicka B, Kruk J (2012) Plastoquinol is more active than α-tocopherol in singlet oxygen scavenging during high light stress of Chlamydomonas reinhardtii, Biochim Biophys Acta: Bioenerg 1817:389–394. Acknowledgements [20] Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes, Meth Enzymol 148C:350–382. only from increased degradation due to ROS [21] Nowicka B, Pluciński B, Kuczyńska P, Kruk J (2016) Physiological characterization of Chlamydomonas reinhardtii acclimated to chronic stress induced by Ag, Cd, Cr, Cu and Hg ions, Ecotoxicol Environ Saf 130:133-145. This work was supported by grant 2013/11/D/NZ1/00303 from the scavenging, but also from inhibition of biosynthesis of National Science Center Poland. these compounds.