Nostoc sp. extract induces oxidative stress-mediated root cell destruction in Mimosa pigra L.
© Sukkhaeng et al.; licensee Springer. 2015
Received: 20 June 2014
Accepted: 15 December 2014
Published: 22 February 2015
Mimosa pigra is an invasive weed in some regions of South East Asia and Australia. Our previous study has revealed that a cyanobacterium, Nostoc sp., extract can inhibit root growth in M. pigra seedlings. In this study, some physiological processes involve oxidative stress-mediated cell death and root ultrastructure were investigated to clarify the mechanisms of root growth suppression and bioherbicidal potential of the extract.
Nostoc sp. extract enhanced overproduction of reactive oxygen species (ROS) at 24 h, the intensity of red fluorescence increased at 72 h, and caused a slightly increased H2O2 consistent with the activation of scavenging enzymes (catalase, ascorbic acid peroxidase, glutathione reductase, and peroxidases). This suggests that oxidative stress occurred in the presence of the extract which was supported by increased cell death and lipid peroxidation at 24 h. Reduction of malondialdehyde content and an increase in cell death at 72 h indicated oxidative damage and cellular leakage. Ultrastructural changes were determined at 72 h by scanning electron micrographs which confirmed the damage of epidermal and root cap cells and the disaggregation and destruction of root tip cells. Transmission electron micrographs showed the dissolution of the middle lamella, deposition of some substances in vacuoles, and abnormal mitochondria (swollen mitochondria and indistinct cristae).
Nostoc sp. extract enhance oxidative stress by ROS production resulting in lipid peroxidation and massive cell death despite the activation of antioxidative enzymes. Understanding mechanism of action of Nostoc sp. extract will provide information for application of the extract to use as natural herbicide for control of M. pigra.
KeywordsNostoc sp Oxidative stress Lipid peroxidation Cell death Root ultrastructure Mimosa pigra
Cyanobacteria are known to produce various kinds of secondary metabolites that can affect many biochemical processes within cells and can influence the growth of surrounding organisms (Leflaive and Ten-Hage ). Natural products from cyanobacteria exhibit various biological inhibitory effects which are cytotoxic, antibacterial, antifungal, and antialgal (Etchegaray et al. ; Kreitlow et al. ; Kulik ). Cyanobacteria have been recognized as options for novel bioactive natural products and use as bio-control agents and herbicides due to their inhibitory activities against some aquatic and terrestrial plants, especially weeds (Duke et al. ; Berry et al. ).
Nostoc sp., a filamentous cyanobacterium (Nostocales), has been reported to produce some chemical substances affecting on various organisms. For example, an alkaloid nostocarboline had an inhibitory activity on Microcystis aeruginosa (Blom et al. ). Nostocyclamide, a cyclic peptide, had antialgal activity and growth inhibitory activities against cyanobacteria and Chlorophyceae (Kobayashi and Kajiyama ). The effect on higher plants was reported by Hirata et al. () that nostocine A produced by Nostoc spongiaeforme TISTR 8169 exhibited an inhibitory activity on root growth of barnyard grass (Echinochloa crus-galli (L.) P. Beauv.). It is possible that Nostoc species has weed suppressing potential. Not much is known about the mechanism of Nostoc extract.
Understanding the mechanism of natural plant compounds is important for research in natural herbicides. Under unfavorable conditions, such as high or low temperature, water deficit, salinity, and some chemical substances, normal metabolisms of plants are disturbed and toxic molecules are produced (Mano ). One of these toxic molecules is ROS which can abstract hydrogen atoms from polyunsaturated fatty acids (the composition of biological membranes), initiate lipid peroxidation, and cause cell death. During this processes, plants suffer from oxidative stress and malondialdehyde (MDA) is produced as a by-product (Halliwell and Chirico ; Gutteridge ). Because of ROS toxicity, plant cells have mechanisms to reduce these toxic compounds by ROS scavenging via antioxidative enzyme systems (Mittler ). Root ultrastructural changes have been used to study the damage of structures and organelles in root tips exposed to some allelochemicals. These include the decreases in the number of ribosome, dictyosome, mitochondria, endoplasmic reticulum, and metabolic products, mitochondrial swelling and loss of cristae, dissolution of the middle lamella, and the irregular-shaped cells (Burgos et al. ; Jiang and Liu ; Yang et al. ).
In our previous study, crude extract from Nostoc sp. was effective on shoot and root growth suppression of M. pigra and had an adverse effect on mitotic cell division (Sukkhaeng et al. ), but the inhibition of cell division might not be the only major mechanism of root growth suppression. The aims of this study were to explore the effect of Nostoc sp. extract on ROS-mediated oxidative stress (superoxide anion radical (O2•−) and lipid peroxidation), ROS metabolism in terms of the alterations of antioxidative enzymes, and oxidative damage (cell death and root ultrastructural changes).
Cyanobacteria culture and extraction
A cyanobacterium, Nostoc sp. (BotKU C3004, voucher specimen is deposited at herbarium of the Department of Botany, Faculty of Science, Kasetsart University) was collected from rice straw in Bangkok, Thailand and isolated by the streak plate method. Nostoc cells were cultured in BG-11 liquid medium (Rippka et al. ), pH 6.5 under daylight fluorescent lamps (300 μmol m−2 s−1) at ambient temperature (32 ± 3°C). The cells in the exponential phase of growth (15 days after culture) were harvested by means of filtration and subsequently dried at 50°C for 72 h. The dried cells were ground to powder and extracted with 80% methanol for 24 h at ambient temperature. The solution was filtered and the supernatant was evaporated in a rotary evaporator to obtain a crude brown gum. The residue was extracted twice and the supernatant was combined with the first one.
The gummy substance was dissolved in water containing 0.1% dimethyl sulfoxide (DMSO) at 0, 0.1, 0.3 and 0.5%. Test solutions of various concentrations (1.5 ml) were added to petri dishes (5 cm in diameter) containing filter paper. Six seeds of M. pigra were placed on the filter paper and kept in the dark at ambient temperature for 24 and 72 h. The water containing 0.1% DMSO was used as a control.
Determination of antioxidative enzyme activities
Three hundred milligrams of frozen roots were crushed to a fine powder in a mortar under liquid nitrogen and homogenize with 3 ml of 25 mM potassium phosphate buffer (pH 7.8) containing 0.4 mM EDTA-4H, 1 mM ascorbic acid, and 2% PVPP. The homogenate was centrifuged at 12,000 rpm for 20 min at 4°C and the supernatant was used as an enzyme extract for the assays of catalase (CAT; EC 188.8.131.52), ascorbic acid peroxidase (APX; EC 184.108.40.206), glutathione reductase (GR; EC 220.127.116.11), and peroxidases (PODs; EC 18.104.22.168) activities.
CAT activity was assayed by determination of the initial rate of hydrogen peroxide disappearance according to Aebi (). Activity was measured spectrophotometrically at 240 nm (E = 0.0394 mM−1 cm−1). GR activity was determined by the method of Halliwell and Foyer (). Activity was determined at 340 nm by measuring the decrease of NADPH absorbance (E = 6.1 mM−1 cm−1). APX activity was assayed by the method of Nakano and Asada (). Activity was determined at 290 nm by measuring the decrease of ascorbic acid (E = 2.8 mM−1 cm−1). PODs activity was determined by the method of Putter (). PODs activity was measured spectrophotometrically at 436 nm by the formation of guaiacol dehydrogenation products (E = 25.5 μM−1 cm−1).
Histochemical detection of ROS
ROS production was estimated using a superoxide anion radical-specific oxidation-sensitive fluorescence dye, dihydroethidium (DHE) (Sunohara and Matsumoto ) with a minor modification. Treated roots were stained with 10 μM DHE in 100 μM CaCl2, pH 4.75, by gently shaking for 2 h at 25°C in the dark. The roots were then soaked in 100 μM CaCl2 for 5 min and observed using a fluorescence microscope (Nikon E600 with a B-2A filter combination, excitation 450–490 nm, emission ≥ 520 nm).
Hydrogen peroxide content was determined according to Velikova et al. (). This method is based on potassium iodide (KI) oxidation by H2O2 to give iodide ion. Iodide reacts with iodine to form triiodide (I3−) which show absorption at 285 nm (Junglee et al. ). For every sample, distilled water was used instead of KI for tissue coloration background. H2O2 standard solutions were prepared for calibration curve.
Determination of root cell death
Treated roots were stained with Evans blue solution (0.025% (w/v) in 100 μM CaCl2, pH 5.6) for 30 min. Stained roots were washed 3 times with 100 μM CaCl2 (pH 5.6), until the dye no longer eluted from the roots (Yamamoto et al. ). Ten root tips were excised (3 mm) and allowed to soak in 200 μl of N,N-dimethylformamide for 24 h. The absorbance of released Evans blue was measured at 600 nm.
Determination of lipid peroxidation
The thiobarbituric acid test, which determines MDA as an end product of lipid peroxidation (Velikova et al. ), was used. The amount of thiobarbituric acid-reactive substances, red pigments, was calculated from the extinction coefficient 155 mM−1 cm−1.
Root ultrastructural studies
For scanning electron microscopy, excised root tips of 5 mm (0 and 0.5% at 72 h treated roots) were prefixed in 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.2), postfixed in 2% osmium tetroxide for 2 h., dehydrated in an acetone series, and dried in critical point dryer (Emitech; K850). After sputter-coating with gold by using an ion coater (Eiko Engineer; IB-2), they were examined with a scanning electron microscope (SEM) (Jeol; JSM5600LV) which operated at 10 kV. For transmission electron microscopy, 3 mm root tip segments were fixed overnight in 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.2). They were postfixed for 2 h in 2% osmium tetroxide in water. The roots were embedded in a Spurr’s resin and polymerized for 7 h at 80°C. Next, 60 nm cross-sliced sections, probably located in the middle part between the root tip and the quiescent center in the root cap, were made on an ultramicrotome (Leica; UCT) and stained with 5% (w/v) uranyl acetate and 2% (w/v) lead citrate. After fixing, the specimens were examined with a transmission electron microscope (TEM) (Jeol; JEM1220) which operated at 80 kV.
All statistical analyses were performed as analysis of variance (ANOVA) and means were compared using Duncan’s multiple range test (DMRT) at p ≤ 0.05.
Results and discussion
Based on our results, we conclude that a Nostoc sp. extract enhanced oxidative stress by ROS production resulting in lipid peroxidation of cell membranes and cell death despite the activation of antioxidative enzymes. This was confirmed by destruction of root tip cells and aberrant mitochondria. These results are consistent with our previous study that the extract inhibited root growth of M. pigra by inducing oxidative stress and cell death. To clarify the mechanism of action of Nostoc sp. extract provides benefit information for application to use as natural herbicide and control M. pigra − invasive weed in some parts of the world. The extract might be developed to use as bioherbicide for weed control. The effects of this extract on other weed species, field experiment, and purification of bioactive compounds need to be investigated.
Ascorbic acid peroxidase
Nicotinamide adenine dinucleotide phosphate
Superoxide anion radical
Reactive oxygen species
Scanning electron microscope
Transmission electron microscope
We are thankful to the Thai Science Scholar Project, Bilateral Research Cooperation (BRC) from Faculty of Sciences, and Kasetsart University Research and Development Institute (KURDI) for financial support. We also thank Scientific Equipment and Research Division, Kasetsart University for using their electron microscope.
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