Nodularin uptake and induction of oxidative stress in spinach (Spinachia oleracea)

Journal of Plant Physiology 168 (2011) 594–600

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Nodularin uptake and induction of oxidative stress in spinach (Spinachia oleracea) Nina Lehtimäki a,1 , Sumathy Shunmugam a,1 , Jouni Jokela b , Matti Wahlsten b , Dalton Carmel a , Mika Keränen a , Kaarina Sivonen b , Eva-Mari Aro a , Yagut Allahverdiyeva a , Paula Mulo a,∗ a b

Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FIN 20014 Turku, Finland Department of Food and Environmental Sciences, University of Helsinki, PO Box 56, Viikki Biocenter, Viikinkaari 9, FIN 00014, Finland

a r t i c l e

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Article history: Received 22 June 2010 Received in revised form 20 September 2010 Accepted 20 September 2010 Keywords: Nodularia Nodularin Oxidative stress Photosynthesis Spinach

a b s t r a c t The bloom-forming cyanobacterium Nodularia spumigena produces toxic compounds, including nodularin, which is known to have adverse effects on various organisms. We monitored the primary effects of nodularin exposure on physiological parameters in Spinachia oleracea. We present the first evidence for the uptake of nodularin by a terrestrial plant, and show that the exposure of spinach to cyanobacterial crude water extract from nodularin-producing strain AV1 results in inhibition of growth and bleaching of the leaves. Despite drastic effects on phenotype and survival, nodularin did not disturb the photosynthetic performance of plants or the structure of the photosynthetic machinery in the chloroplast thylakoid membrane. Nevertheless, the nodularin-exposed plants suffered from oxidative stress, as evidenced by a high level of oxidative modifications targeted to various proteins, altered levels of enzymes involved in scavenging of reactive oxygen species (ROS), and increased levels of ␣-tocopherol, which is an important antioxidant. Moreover, the high level of cytochrome oxidase (COX II), a typical marker for mitochondrial respiratory protein complexes, suggests that the respiratory capacity is increased in the leaves of nodularin-exposed plants. Actively respiring plant mitochondria, in turn, may produce ROS at high rates. Although the accumulation of ROS and induction of the ROS scavenging network enable the survival of the plant upon toxin exposure, the upregulation of the enzymatic defense system is likely to increase energetic costs, reducing growth and the ultimate fitness of the plants. © 2010 Elsevier GmbH. All rights reserved.

Introduction Cyanobacteria are a large group of oxygenic photosynthetic bacteria that are present in a wide range of habitats. The occurrence of cyanobacteria in fresh and brackish water often results in surface blooms, which may lead to a release of nutrients accelerating eutrophication as well as to a release of wide range of toxic or bioactive compounds having adverse effects on the faunae (Codd, 1995). Recent studies have demonstrated that certain cyanotoxins may disturb the function of plants as well; for example, microcystin retards the growth of seedlings of a variety of crop plants by inhibiting protein phosphatases and photosynthesis (MacKintosh et al., 1990; Babica et al., 2006). In many organisms, cyanotoxins induce oxidative stress and accumulation of reactive oxygen species (ROS) (Pflugmacher et al., 2007a,b), which may damage cellular compo-

Abbreviations: APX, ascorbate peroxidase; COX II, cytochrome oxidase II; FW, fresh weight; PRXQ, peroxiredoxin Q; PS, photosystem; PTOX, plastid terminal oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase. ∗ Corresponding author at: Molecular Plant Biology, Department of Biochemistry and Food Chemistry, Biocity A, FIN 20520, University of Turku, Finland. Tel.: +358 2 3338076; fax: +358 2 3338075. E-mail address: pmulo@utu.fi (P. Mulo). 1 These two authors have made equal contributions. 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.09.013

nents such as DNA, membranes and proteins. The harmful effects of oxidative stress are prevented or reduced by the action of an elaborate ROS scavenging network, which consists of enzymatic components (such as superoxide dismutase (SOD), catalase, glutathione and ascorbate peroxidases and glutathione reductase) and various antioxidants (glutathione, ascorbate and tocopherol) (Apel and Hirt, 2004; Møller et al., 2007). Cyanotoxins are classified into hepatotoxins (microcystin, nodularin and cylindrospermopsin), neurotoxins (anatoxin-a, anatoxin-a(S) and saxitoxins) and irritant-dermal toxins according to the symptoms they induce in humans and vertebrates. Among cyanotoxins, microcystin produced by Microcystis sp. is a common and well studied toxin. It has been shown that, in addition to its toxic effects on animals, microcystin exposure may also result in oxidative stress in plants, which leads to necrosis of the leaves and stunted growth of the plants (Järvenpää et al., 2007; Peuthert et al., 2007). Another important but less studied cyanotoxin is nodularin, produced by nitrogen-fixing Nodularia spumigena, which is the most common cyanobacterium species found in the Baltic Sea (Sivonen et al., 1989). Additionally, Nodularia sp. has been found in various freshwater lakes (Beattie et al., 2000). Nodularin is a cyclic pentapeptide, hepatotoxin and tumor promoter (Ohta et al., 1994). Exposure to nodularin induces oxidative stress in mussels (Davies et al., 2005; Kankaanpää et al., 2007), fish (Vuorinen et al.,

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2009) and marine macroalga Fucus vesiculosus (Pflugmacher et al., 2007b). Plants growing in the shore zone of the Baltic Sea, or by the lakes occupied by Nodularia sp., are occasionally exposed to cyanobacteria-contaminated water, which may disturb the growth of plants, while agricultural crop plants may be particularly contaminated with cyanotoxins when irrigated with surface waters containing cyanobacterial blooms. It is conceivable that the plants can accumulate toxins in the leaves, roots and other organs, which may cause deleterious effects not only to the plants, but also to the heterotrophs fed by contaminated plants. In the present study, we explored the primary physiological target(s) of nodularin on the metabolic pathways of Spinachia oleracea, which is an important model organism in plant biochemistry and photosynthesis research. Understanding the impact of nodularin on plant metabolism at the molecular level will allow further studies to determine ecological effects of nodularin exposure to the surrounding ecosystem. Material and methods Cyanobacterial and plant material Nodularin-producing (AV1) and non-nodularin-producing (HKVV) Nodularia sp. cells (University of Helsinki Culture Collection (UHCC), Division of Microbiology, Department of Food and Environmental Sciences) were grown under continuous light of 20 ␮mol photons m−2 s−1 in Z8 medium with salt and without added nitrogen at 25 ◦ C (Lehtimäki et al., 1997). The cells were harvested after 4 weeks growth in their stationary phase, pelleted and washed with tap water. Thereafter, the cell pellets were weighed, frozen and thawed several times to break the cells and release the toxins. Just before use, the pellets were suspended in tap water. Spinach seeds (Spinachia oleracea L. cv. Nores) were purchased from Nelsons OY (Finland). The seeds were sown on vermiculite, and the seedlings were transferred after germination to the pots filled with commercial soil (Kekkilä, Finland). The spinach plants were grown under 250 ␮mol photons m−2 s−1 , an 8 h photoperiod and a temperature of 23 ◦ C in three distinct groups. Each group was composed of eight plants grown in two separate pots. During the growth period of five weeks, one of the groups was watered daily with tap water, and for the second group, the tap water was supplemented twice a week with 0.25 g fresh weight (FW) of Nodularia HKVV cell extract (dissolved in 30 mL of tap water) per plant. The third group was watered with tap water supplemented twice a week with 0.25 g fresh weight (FW) of Nodularia AV1 cell extract (dissolved in 30 mL of tap water) per plant (0.34 ␮M nodularin), which was the dose resulting in severe visible symptoms during the growth period of five weeks. Cyanobacterial extracts were provided by watering the bottom of the soil and care was taken not to contaminate the surface of the plants. Because the focus was on the primary effects of nodularin, the metabolism of young, healthy-looking leaves of the nodularintreated plants was studied, and the small, dark green leaves were harvested for the experiments. The metabolism of the old, but still vital, leaves with clear visible symptoms (chlorotic spots) was studied in order to detect later steps of intoxication, while the completely bleached leaves were excluded from the analyses. For comparison, leaves of corresponding age were studied in the control and HKVV-treated plants.

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shaking in 15 mL plastic tubes with 70% methanol in 80 ◦ C. Prior to HPLC injection, methanol extracts were filtered through 0.2 ␮m filters. LC–MS analyses of extracts were performed with an Agilent 1100 Series LC/MSD Ion Trap XCT Plus System (Agilent Technologies, Palo Alto, CA) using a Phenomenex Luna C18(2) (150 × 2.0 mm, 5 ␮m, Phenomenex, Torrance, CA) LC-column. The mobile phase was composed of 0.1% formic acid in water (A) and 2-propanol (B). The gradient run was from 20% B to 70% (B) over 20 min and then a 9 min wash with 100% (B) at a flow rate of 0.15 mL min−1 at 40 ◦ C using 5 or 10 ␮l injections. Nodularin was detected with a diode array detector (238 nm) and with MS using electrospray ionization (ESI) set in positive ion mode. The nebulizer gas (N2 ) pressure was 35 psi (240 kPa), desolvation gas flow rate 8 L min−1 and the desolvation temperature 350 ◦ C. The capillary voltage was set to 5000 V, the capillary exit offset was 300 V, the skimmer potential was 66 V and the trap drive value was 73. Spectra were recorded at a scanning range of 100–850 m/z and rate of 26,000 m/z s−1 . Product ion spectra using auto MS mode with precursor ion m/z 825.5 was recorded and nodularin quantification was based to the integration of the nodularin-specific product ion peak m/z 389.3 from the extracted ion chromatogram. Eight reference nodularin (gift from Zbigniew Grzonka, Faculty of Chemistry, University of ´ Gdansk, Poland) solutions from 0.5 to 9000 ng/mL 70% methanol were prepared. The linear response curve (R2 = 0.99996) for calculating nodularin concentrations in extracts and further nodularin amounts in solid samples was obtained. Germination test For germination tests, 100 spinach seeds were sown on vermiculite and watering of plants with cyanobacterial extracts (0.5 g FW) was started immediately. After two weeks of treatment, the number of germinated seeds from each treatment was counted. Pigment and ˛-tocopherol analysis Pigments (chlorophyll a and b, neoxanthin, violaxanthin, lutein and ␤-carotene) and ␣-tocopherol were extracted from leaf discs (diameter 5 mm) with 300 ␮L of pure methanol. After centrifugation and filtration of the extracts, photosynthetic pigments were separated by HPLC according to Gilmore and Yamamoto (1991) with a reverse phase C18 column (LiChroCART 125-4, Hewlet Packard), series 1100 HPLC device with diode array and fluorescence detector (Agilent Technologies, Palo Alto, CA). Buffer A consisted of acetonitrile–methanol–Tris–HCl buffer 0.1 M pH 8.0 (72:8:3, v/v) and buffer B consisted of methanol–hexane (4:1, v/v). A constant flow rate 0.5 L min−1 was used. The program started with an isocratic run with buffer A for 4 min followed by a linear gradient for 15 min from 0% buffer B to 100% buffer B. The isocratic run of buffer B lasted 26 min. The column was re-equilibrated between samples for a minimum of 10 min with buffer A. Pigments were detected at 440 nm, and ␣-tocopherol by fluorescence (ex = 295 nm, em = 340 nm). Pigment standards were supplied from DHI Lab Products and the ␣-tocopherol was supplied from Sigma–Aldrich. Determination of chlorophyll content The chlorophyll content from isolated thylakoid membranes was determined as described in Porra et al. (1989).

Quantitation of nodularin

Photosynthetic activity

Nodularin content of the roots and leaves of spinach and soil as well as cyanobacterial water extracts was quantified. Weighted (wet weight) roots, leaves and soil were treated for 60 min without

Oxygen evolution To measure the Photosystem (PS) II oxygen-evolving capacity, the thylakoid membranes were isolated and electron transfer

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activity of PSII from water to DCBQ (2,6-dichloro-p-benzoquinone, 1 mM) was measured as oxygen evolution under red saturating light using a Hansatech oxygen electrode. Ten ␮g of chlorophyll was used for each measurement. Oxido-reduction of P700 P700 redox changes were measured from leaf discs with JTS-10 (Biologic, France). P700 was oxidized by a far-red LED (720 nm), and computed as I/I700 = I/I820 nm − 0.8 × I/I880 nm . Isolation of proteins The leaves were frozen in liquid nitrogen and homogenized in a shock buffer (10 mM HEPES-KOH, pH 7.6, 5 mM sucrose, 5 mM MgCl2 ), and the homogenate was filtered through miracloth and centrifuged at 4000 × g for 4 min at 4 ◦ C. The supernatant was collected and the thylakoid pellet was resuspended in storage buffer (10 mM HEPES-KOH pH 7.5, 100 mM sucrose, 5 mM NaCl, 10 mM MgCl2 ). The protein content of the fractions was measured as described by Bradford (1976). SDS-PAGE and immunoblotting The proteins were loaded on gels and separated by SDSPAGE (14% acrylamide, 6 M urea) according to Laemmli (1970). After electrophoresis, the proteins were electroblotted to a polyvinylidene fluoride (PVDF) membrane (Millipore, http://www.millipore.com/). The antibodies against PSAB, ATPase, SOD, PRXQ, APX isoforms and COXII were purchased from Agrisera (Vännäs, Sweden), the antibody against the D1 protein from Research Genetics (Huntsville, AL; Kettunen et al., 1996), and the antibodies against CP43 and CP47 were gifts from Prof. R. Barbato, and PTOX from Dr. M. Kuntz. The linearity of each antibody was first tested for spinach samples, and thereafter the gels were loaded in the linear range according to the test (25 ␮g proteins for D1, CP47 and SOD; 5 ␮g for CP43 and PSAB; 10 ␮g for COX II; 50 ␮g for PTOX, PRXQ, ATPase and APX). Oxidative modifications (i.e. carbonylation) of the thylakoid proteins were studied using the OxyBlotTM Protein Oxidation Detection Kit (Chemicon International, Temecula, CA, USA). Immunoblots were quantified with a Fluorchem Image Analyzer (Alpha Innotech Corporation). APX activity and determination of ascorbate content APX activity extracted from fresh leaves was determined according to Foyer et al. (1989) with the modifications of Pätsikkä et al. (2002). Ascorbate was extracted from the leaves and ascorbate content was assayed according to Foyer et al. (1983).

Statistical analysis The numerical data were subjected to statistical analysis using the Student’s t-test, with statistical significance at the level of P < 0.05. Results Phenotype of the plants treated with cyanobacterial extracts Watering of spinach seedlings with crude water extract from the nodularin-producing cyanobacterium Nodularia spumigena had distinct effects on the plant phenotype. The leaves of the control plants were dark green and exuberant, whereas the leaves of the plants watered with the extract of N. spumigena AV1 suffered from severe chlorosis (Fig. 1). The most severe symptoms were detected in the oldest leaves, which were partly colorless, while the youngest leaves looked dark green and quite healthy. After six weeks of growth, the oldest leaves died. In contrast, the treatment of plants with water extract from non-nodularin producing Nodularia HKVV did not result in necrosis and death of the leaves during the time course of the experiment. HPLC analysis of pigments corroborated the visual observations: yellowish parts of the spinach leaves of nodularin-exposed plants possessed ca. 50% less Chl a and Chl b than those of the control plants, whereas the chlorophyll content of the HKVV-exposed plants was similar to that of the control (Fig. 2(A)). It must be emphasized, however, that the chlorophyll content of the green parts of the leaves from nodularin-exposed plants did not differ from that of the control leaves (Fig. 2(A)). The Chl a/b ratio of the green parts of all the plants was 3.1–3.2, but a somewhat lower chlorophyll a/b ratio (2.8) was detected in the yellow parts of the leaves from the nodularin-exposed plants. Similarly, no distinct differences in the content of neoxanthin, lutein, ␤-carotene and violaxanthin could be detected between the green parts of the leaves from the nodularin-exposed plants and the leaves of the control or HKVV-exposed plants (Fig. 2(B)). Notably, however, the yellow parts of the leaves from the nodularin-exposed plants always contained markedly less chlorophyll and carotenoid pigments than the control or HKVV-exposed plants (Fig. 2). Intriguingly, ␣-tocopherol content in both green and yellow leaves of the plants treated with nodularin-containing extract was ca. 1.3-fold higher than in the control (Fig. 2(B)). To test whether the adverse effect of the nodularin-containing extract could be detected already at the stage of germination, a fixed number of spinach seeds was sowed on vermiculite, doused with water, nodularin-containing AV1 or non-nodularincontaining HKVV cyanobacterial extracts, and the efficiency of germination was examined. Interestingly, germination was not

Fig. 1. Phenotype of the control spinach plants, and plants watered with non-nodularin-containing and nodularin-containing Nodularia extracts. The plants were grown for five weeks, and watered with cyanobacterial crude extracts (0.25 g FW/plant) twice a week. “HKVV extract” denotes plants exposed to non-nodularin-containing, and “AV1 extract” denotes nodularin-containing extract of Nodularia sp.

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Photosynthetic machinery of spinach plants exposed to cyanobacterial extracts

Fig. 2. Pigment and ␣-tocopherol content of the control spinach plants, and plants watered with non-nodularin-containing and nodularin-containing Nodularia extracts studied by HPLC. Pigments were detected at 445 nm, and ␣-tocopherol by fluorescence (ex = 295 nm, em = 340 nm). Young, green leaves and old yellowish leaves of the nodularin-exposed plants were analyzed separately. (A) Chlorophyll a and b content of the control spinach plants, and plants watered with nonnodularin-containing and nodularin-containing Nodularia extracts. (B) Carotenoid and ␣-tocopherol content of the control spinach plants, and plants watered with non-nodularin-containing and nodularin-containing Nodularia extracts. “HKVV extract” denotes plants exposed to non-nodularin-containing, and “AV1 extract” denotes nodularin-containing extract of Nodularia sp. Data is given as average ± S.D., n = 4. The asterisk denotes a statistically significant difference from the control.

affected by watering the seeds with cyanobacterial extracts (Fig. 3). Nodularin uptake in spinach To determine whether nodularin can enter the plants via the root system, the amount of nodularin was determined by HPLC analysis. A significant amount of nodularin could always be detected in the old leaves of the nodularin-exposed plants (66 ± 5.1 pmol/g FW, n = 3), whereas the nodularin content of young leaves and roots varied (from 0 to 30 pmol/g FW in the young leaves and from 0 to 24 pmol/g FW in the roots). No nodularin was detected in the leaves of the control or HKVV-exposed plants. A significant amount of nodularin remained intact in the soil and was therefore not taken up by the plants (data not shown).

Possible effects of nodularin on the photosynthetic machinery of spinach leaves were first studied by performing protein blot analysis of key photosynthetic proteins. No marked difference in the content of PSII (D1, CP43 or CP47) and PSI (PSAB) proteins was detected between the control plants and the plants treated with cyanobacterial extracts, even if the old yellowish leaves of the plants exposed to nodularin containing extract were examined (Fig. 4(A)). Also the content of ATPase did not vary between the differently-treated plants (Fig. 4(A)). Likewise, no differences were detected in the PSII capacity between the spinach plants when oxygen evolution activity of the isolated thylakoids was measured either from the young, healthy leaves or from old, yellowish leaves by Hansatech oxygen electrode using DCBQ as an artificial electron acceptor (Fig. 4(B)). However, the rate of re-reduction of P700 + , the primary donor of PSI, in the dark was slightly faster in the nodularin-exposed plants than in the control or HKVV-exposed plants (Fig. 4(C)). ROS scavenging network in spinach plants exposed to cyanobacterial extracts Since the nodularin-exposed plants suffered from chlorosis, which is often one of the symptoms of oxidative stress, we next focused on oxidative modifications of spinach proteins. Various membrane and soluble proteins in the nodularin-exposed plants showed markedly more oxidative damage than those of the control or HKVV-exposed plants (Fig. 5(A)). Moreover, the content of ascorbate peroxidase (APX) enzymes, which scavenge hydrogen peroxide by using reduced ascorbate as an electron donor, was modified not only in the nodularin-exposed plants, but also in the HKVV-treated plants (Fig. 5(B)). The content of thylakoid-bound APX (tAPX) and stromal APX (sAPX) was markedly decreased (Fig. 5(B)), whereas less drastic changes in the level of cytoplasmic APX (cAPX) and SOD were detected (Fig. 5(B)). Peroxiredoxin Q (PRXQ) content of the nodularin-exposed plants did not differ significantly from that of the control, while the level of PRXQ in the HKVV-exposed plants was lower than in the control (Fig. 5(B)). Despite obvious changes in the oxidative status, no significant difference in the ratio of reduced to oxidized ascorbate were detected between the differentially-treated spinach plants (Fig. 5(C)). Accordingly, although the activity of APX enzyme was somewhat lower in the plants treated with cyanobacterial extracts as compared to the control, the difference was not statistically significant (Fig. 5(D)). Since mitochondria are another potential source of ROS, we examined the level of mitochondrial cytochrome oxidase II (COX II). The amount of cytochrome oxidase is known to be directly proportional to respiratory activity, at least in Chlamydomonas reinhardtii (Petroutsos et al., 2009), and indeed the amount of COX II in the nodularin-exposed plants was significantly higher than in the control or in the HKVV-exposed plants (Fig. 5(B)). The level of plastid terminal oxidase, which is the key player in chlororespiration (Kuntz, 2004) did not differ significantly among the control, nodularin-exposed and HKVV-exposed plants (Fig. 5(B)). Discussion

Fig. 3. Germination of the seeds treated with water, non-nodularin-containing and nodularin-containing Nodularia extracts. One hundred spinach seeds were sown on vermiculite, doused with water, non-nodularin-containing or nodularin-containing Nodularia extract, and the number of germinated seeds was counted after two weeks. Data is given as average ± S.D., n = 4, no statistically significant differences between the treatments were detected.

Nodularin exposure results in bleaching and reduced growth of spinach Cyanobacterial secondary metabolites have been studied intensively during the past decade due to increased mass occurrences

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Fig. 5. Oxidative status of spinach plants treated with water, non-nodularincontaining and nodularin-containing Nodularia extracts was studied after four weeks treatment. (A) Steady-state level of carbonylated spinach proteins using the OxyblotTM Protein Oxidation Detection Kit. 15 ␮g of protein was loaded in each well. (B) Levels of the SOD, PRXQ, cAPX, sAPX, tAPX, PTOX and COX II proteins in the spinach leaves. After SDS-PAGE, proteins were electroblotted on a PVDF membrane and probed with the respective antisera according to the linearity tests. Percentages denote average from 2 to 3 biological replicates, the SD was never more than 20% of the values. (C) The ratio of reduced ascorbate (Asc) to dehydroascorbate (DHA) in spinach leaves. The acid-extracted ascorbate content of the leaves was measured spectrophotometrically at 265 nm. (D) Ascorbate peroxidase (APX) activity in the spinach leaves. Spectrophotometric quantification of APX activity was performed at 265 nm from total leaf extracts. In all experiments, old, yellowish leaves were used in the experiments, three to ten independent measurements were performed and the values are means ± SD.

Fig. 4. Properties of photosynthetic machinery. Photosynthetic properties and the protein content of the photosynthetic machinery of the control spinach plants, and plants watered with non-nodularin-containing and nodularin-containing Nodularia extracts were studied after five weeks treatment. (A) Thylakoid protein content of the old spinach leaves. After SDS-PAGE, proteins were electroblotted on a PVDF membrane and probed with the D1, CP43, CP47, PSAB and ATPase antisera. Samples were loaded on gels on protein basis according to the linearity tests. Percentages denote average from 2 to 3 biological replicates. (B) Oxygen-evolution rates of the spinach thylakoid membranes isolated from young and old leaves, as indicated, with DCBQ as an electron acceptor. Data is given as average ± S.D., n = 3, no statistically significant differences between the treatments were detected. (C) Dark re-reduction of P700 + in the old leaves of control, AV1-exposed and HKVV-exposed spinach plants. P700 was oxidized by far red light for 30 s and P700 + re-reduction was monitored in darkness. Curves were normalized to the maximal signal, a representative curve is shown. a.u. denotes for arbitrary units. “HKVV extract” denotes plants exposed to non-nodularin-containing, and AV1 extract denotes nodularin-containing extract of Nodularia sp.

or blooms with deleterious consequences for the environment. In particular, the effects of microcystin on aquatic ecosystems as well as on terrestrial plants and animals have been widely examined, whereas the physiological and ecological consequences of nodularin exposure are not yet well understood. Here, we have shown that the long-term watering of terrestrial plants with nodularincontaining N. spumigena extract results in uptake of nodularin in the leaves and roots, which in turn leads to severe chlorosis and reduced growth of the plants (Fig. 1). Although accumulation of only nodularin was studied here, it must be emphasized that in addition to nodularin, various other potentially harmful cyanobacterial components (such as other non-ribosomal peptides like spumigins and nodulapeptins acting as protease inhibitors) are also found in the N. spumigena AV1 extract (Fuji et al., 1997; Fewer et al., 2009). In accordance with the accumulated amount of nodularin, the older leaves of the nodularin-exposed plants showed more severe symptoms than the young leaves (Fig. 1). The visual symptoms were reflected at the molecular level, evidenced by less chlorophyll and carotenoids accumulating in the yellow parts of the leaves from the nodularin-exposed plants than in the control (Fig. 3), while no difference was detected between the green parts of the leaves from

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the nodularin-exposed plants and the control and HKVV-exposed plants. Similar bleaching of the leaves has been detected in plants treated with microcystin (Pflugmacher et al., 2007a). Notably, the germination of spinach seeds was not inhibited by nodularincontaining extract, so the deleterious effect on the growth was evident only during the later phases of plant development. Microcystin treatment has resulted in varying effects on germination in different plant species: microcystin inhibited the germination of Brassica napus seeds, whereas no effects were seen on germination of Oryza sativa (Chen et al., 2004). Nodularin does not markedly damage the photosynthetic machinery of spinach Despite serious phenotypic effects of nodularin treatment, the photosynthetic performance of the nodularin-exposed plants did not differ from that of the control (Fig. 4). No differences were detected in the rate of PSII oxygen evolution either in the young or in the old leaves, indicating that the PSII in the plants exposed to nodularin-containing extract is fully functional, and hence PSII does not seem to be the primary target of nodularin (Fig. 4(B)). Accordingly, the photosynthetic apparatus of the nodularin-exposed leaves was intact, as deduced from unchanged levels of photosynthetic proteins in the thylakoid membranes (Fig. 4(A)). Thus, although the amount of functional photosystems in the completely yellow leaves of the nodularin-exposed plants is likely to be very low, the function of remaining photosystems is not affected by the nodularin exposure. Controversial results have been obtained from various studies concerning the effects of microcystin on photosynthesis. It has been shown in many studies that microcystin inhibits the photosynthetic activity of terrestrial and aquatic plants as well as algae (Babica et al., 2006), while Järvenpää et al. (2007) have reported that longterm microcystin exposure of Brassica oleracea and Sinapis alba did not have any effect on FV /FM reflecting the capacity of PSII. Such results may arise from varying dosages of the toxin used, differences in the uptake efficiency, in the detoxification mechanisms and in the sensitivity of an individual species to a specific toxin. In line with the results demonstrating that the photosynthetic machinery of the nodularin-exposed leaves is functional, no drastic differences in the content of reduced and oxidized ascorbate or in the total activity of APX enzymes were detected (Fig. 5). However, the content of total APX was decreased in the nodularin-exposed as well as HKVV-exposed leaves, indicating that chloroplasts are able to adjust the function of their antioxidant network according to environmental cues and keep the photosynthetic machinery functional under the toxin treatment. The only photosynthetic parameter found to be changed in the plants exposed to nodularincontaining extract was the rate of P700 + re-reduction (Fig. 4(C)), which has been interpreted to represent the activity of cyclic electron transfer around PSI. Cyclic electron transfer, which changes the ATP/NADPH ratio in the chloroplasts, has been shown to be activated upon stress conditions, such as low ambient CO2 concentration and drought. Thus, activation of cyclic electron transfer may balance the modulated energy requirements of the plants. Nodularin induces oxidative stress in spinach Although the photosynthetic metabolism of the nodularinexposed plants remained intact, the leaves clearly suffered from oxidative stress, as illustrated by the phenotype of the plants (Fig. 1), reduced pigment content (Fig. 2), oxidative modifications of the proteins (Fig. 5), altered level of ROS scavenging enzymes (Fig. 5), and the increased level of ␣-tocopherol (Fig. 2). Since mitochondria are a potential source of ROS in plant cells, preliminary insight into the respiratory electron transfer chain in

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mitochondria was taken. Indeed, the high level of COX II, a typical marker for respiratory protein complexes (Petroutsos et al., 2009), suggests that the respiratory capacity is increased in the nodularinexposed plants. Actively respiring plant mitochondria produce ROS at high rates (Møller, 2001). Moreover, oxidative stress is known to increase respiratory activity in Arabidopsis thaliana (Tiwari et al., 2002), and indeed, our preliminary experiments show that oxygen consumption in crude extracts of nodularin-exposed spinach leaves was higher than in the controls (data not shown). In line with these results, various recent studies focusing on different organisms have evidenced induction of oxidative stress upon exposure to nodularin (Davies et al., 2005; Kankaanpää et al., 2007; Persson et al., 2009). Similarly to spinach, exposure of a brown alga Fucus vesiculosus to nodularin led to marked increases in the activity of lipid peroxidation as well as many ROS scavenging enzymes (Pflugmacher et al., 2007a,b) indicating that promotion of oxidative stress is a common denominator in the response of higher plants and algae to exposure of cyanotoxins. In the present study, we show that the exposure of plants to a non-toxic cyanobacterial extract also leads to changes in the antioxidative network, implying involvement of not only nodularin, but also other, thus far uncharacterized cyanobacterial compounds. However, the most drastic changes were detected only in the phenotype and metabolism of the nodularin-exposed plants, which indicates that nodularin per se is the main determinant for the damage. Although accumulation of ROS and induction of the ROS scavenging network enable survival of the plant upon toxin exposure, alteration of the enzymatic defense system may increase energetic costs, reducing the growth and the ultimate fitness of plants. Acknowledgements This work was financially supported by the Academy of Finland (130075 and 118637), Turku University Foundation and Nordic BioH2 Program (06-Hydr-C13). Y.A. acknowledges the Kone Foundation. References Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 2004;55:373–99. Babica P, Blaha L, Marsalek B. Exploring the natural role of microcystins – a review of effects on photoautotrophic organisms. J Phycol 2006;42:9–20. Beattie KA, Kaya K, Codd GA. The cyanobacterium Nodularia PCC 7804, of freshwater origin, produces [L-Har2 ]nodularin. Phytochemistry 2000;54:57– 61. Bradford MM. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal Biochem 1976;72:248–54. Chen J, Song L, Dai J, Gan N, Liu Z. Effects of microcystins on the growth and the activity of superoxide dismutase and peroxide of rape (Brassica napus L.) and rice (Oryza sativa L.). Toxicon 2004;43:393–400. Codd GA. Cyanobacterial toxins: occurrence, properties and biological significance. Water Sci Technol 1995;32:149–56. Davies WR, Siu WHL, Jack RW, Wu RSS, Lam PKS, Nugegoda D. Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis). Mar Pollut Bull 2005;51:1026–33. Fewer DP, Jokela J, Rouhiainen L, Wahlsten M, Koskenniemi K, Stal LJ, et al. The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena. Mol Microbiol 2009;73:924–37. Foyer C, Rowell J, Walker D. Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 1983;157:239–44. Foyer CH, Dujardyn M, Lemoine Y. Responses of photosynthesis and the xanthophyll and ascorbate–glutathione cycles to changes in irradiance, photoinhibition and recovery. Plant Physiol Biochem 1989;27:751–60. Fuji K, Sivonen K, Adachi K, Noguchi K, Sano H, Hirayama K, et al. Comparative study of toxic and non-toxic cyanobacterial products: novel peptides from toxic Nodularia spumigena AV1. Tetrahedron Lett 1997;38:5525–8. Gilmore AM, Yamamoto HY. Resolution of lutein and zeaxanthin using a non-endcapped, lightly carbon-loaded C18 high-performance liquid chromatographic column. J Chromatogr 1991;543:137–45.

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