Pollutant-induced cell death and reactive oxygen species accumulation in the aerial roots of Chinese banyan (Ficus microcarpa)

Industrial pollutants induce the production of toxic reactive oxygen species (ROS) such as O2.−, H2O2, and ·OH in plants, but they have not been well quantified or localized in tissues and cells. This study evaluated the pollutant- (HSO3−, NH4NO3, Al3+, Zn2+, and Fe2+) induced toxic effects of ROS on the aerial roots of Chinese banyan (Ficus microcarpa). Root cell viability was greatly reduced by treatment with 20 mM NaHSO3, 20 mM NH4NO3, 0.2 mM AlCl3, 0.2 mM ZnSO4, or 0.2 mM FeSO4. Biochemical assay and histochemical localization showed that O2.− accumulated in roots in response to pollutants, except that the staining of O2.− under NaHSO3 treatment was not detective. Cytochemical localization further indicated that the generated O2.− was present mainly in the root cortex, and pith cells, especially in NH4NO3- and FeSO4-treated roots. The pollutants also caused greatly accumulated H2O2 and ·OH in aerial roots, which finally resulted in lipid peroxidation as indicated by increased malondialdehyde contents. We conclude that the F. microcarpa aerial roots are sensitive to pollutant-induced ROS and that the histochemical localization of O2.− via nitrotetrazolium blue chloride staining is not effective for detecting the effects of HSO3− treatment because of the treatment’s bleaching effect.

Scientific RepoRts | 6:36276 | DOI: 10.1038/srep36276 oxidative stress to plants. NO can rapidly react with O 2 .− to form ONOO − , which may transform to · OH, the most reactive and toxic ROS 20,21 . Moreover, in the presence of nitrate (NO 3 − ) assimilation, · OH can be generated and cause free radical-induced injury 22 . Zn and Fe are redox-active metals, and redox cycling catalyses the production of ROS through the Fenton reaction or the peroxidase-catalysed reaction in the presence of O 2 and NADH 23 . As a redox-inactive metal, Al cannot directly participate in biological redox reactions with oxygen, but it can inhibit antioxidant enzymes, causing the accumulation of ROS in cells. ROS can cause cell death and organ senescence, because they readily participate in chain reactions between free radicals and membrane lipids and proteins, resulting in the breakdown of membranes, disturbance of mitosis, inhibition of DNA synthesis, and inactivation of enzymes 4,20,24 .
The deposition of atmospheric sulfur, nitrogen, and industrial dust containing metals has caused the decline of indigenous tree species in South China, but the mechanisms are incompletely understood 5,6,25 . Our previous studies indicated that different forms of pollutants, alone or in combinations, are involved in accelerating oxidative process, causing decreased rates of electron transport and damaged membrane systems in leaf cells 2,5,6 . The toxic effects of ROS caused by various pollutants on aerial roots, however, have been rarely investigated or compared 18 . Aerial roots directly contact air and soil pollutants, and their growth was found to be restricted in industrially polluted regions in subtropical China 26 . In the current study, we evaluated the oxidative stress induced by various industrial pollutants in the aerial roots of Chinese banyan. Chinese banyan is a common landscape tree with a unique aerial root system that grows downward along the trunk to the soil 27 . We also compare methods for quantifying ROS.

Methods
Plant material and pollutants. Chinese banyan, Ficus microcarpa Linn. f. (Moraceae), is a native evergreen tree that is used for urban greening in South China 27 . In April 2015, newly sprouted aerial roots were removed from 15 mature trees growing in the South China Botanical Garden, Guangzhou, China. Each aerial root segment was 5 cm long had a root tip on one end. The root segments were quickly transferred to the laboratory and rinsed with distilled water and then wiped dry.
The aerial root samples (6-8 for per tree from 15 trees) were vacuum-infiltrated for 30 min with distilled water (control, pH 6.09), 20 mM NaHSO 3 (pH 3.08), 20 mM NH 4 NO 3 (pH 4.86), 0.2 mM AlCl 3 (pH 4.07), 0.2 mM ZnSO 4 (pH 5.48), or 0.2 mM FeSO 4 (pH 4.47). Vacuum infiltration was used in order to decrease the differences in the penetration rates of the different ions into the root segments and shorten the treatment period. We referred the atmospheric sulfur and nitrogen, and surface soil metal concentrations in industrially polluted site in South China as background information 2,5,18,26,28 . Based on these reported and our preliminary data, we treated our aerial root samples by designated pollutant concentrations as mentioned above. During treatment, the root samples were kept in an incubator (10 h light and 14 h dark) at 25 °C. A subsample of each root sample (a 3-cm length from the root tip) was then used to determine aerial root viability, the histochemical localization of ROS, and ROS content as described in the following sections.
Aerial root viability. Aerial root viability was determined by Evans blue staining 29 . The 3-cm-long subsamples (five per treatment) were immersed in a 0.25% solution of Evans blue (E2129, Sigma); after 12 h, the subsamples were washed with distilled water to remove the Evans blue solution from the root surface. The dyed root samples were photographed with a digital camera (DSC-F717, Sony, Japan) and then chopped into small pieces and placed in a 1% sodium dodecyl sulfate (SDS) solution for 24 h to completely extract the blue stain. The blue extract, which represented dead cells, was quantified with a spectrophotometer (Lambda 650, Perkin-Elmer, USA) at 600 nm.

Histochemical and cytochemical localization of O 2
.− . O 2 .− was localized by staining with nitrotetrazolium blue chloride (NBT, N6876, Sigma) 18 . The 3-cm-long subsamples (five per treatment) were immersed in HEPES-NaOH buffer (pH 7.6) containing 0.5 mg of NBT/ml and 10 mM NaN 3 . The subsamples were vacuum infiltrated in this NBT solution for 30 min and were then held at room temperature until the blue colour (NBT-O 2 .− ) became visible. The NBT-stained roots were photographed with a digital camera (DSC-F717, Sony, Japan) before semi-thin transverse sections (8 μ m thick) were prepared. Semi-thin section was conducted by fixing aerial root samples in 0.1 M phosphate buffer (pH 7.2) containing 2% glutaraldehyde and 2.5% Paraformaldehyde. After 6 times wash with 0.1 M phosphate buffer, they were dehydrated by alcohol steeply and eddied in flat molds using EPON812 resin. Sctions (2 μ m) were cut by ultramicrotome (Leica, UC6, Germany). The sections were observed and photographed with a light microscope (AX70, Olympus, Japan) and a digital camera (DP50, Olympus, Japan).
Histochemical localization of H 2 O 2 . H 2 O 2 was localized by staining with 3,3′ ,5,5′ -Tetramerthyl benzidine dihydrochloride hydrate (TMB, V900355, Sigma) 30 . The 3-cm-long subsamples (five per treatment) were immersed in 10 mM sodium-citrate buffer (pH 4.0) containing 1 mM TMB at room temperature until the TMB-H 2 O 2 formazan became visible. The stained roots were then photographed with a digital camera (DSC-F717, Sony, Japan). · OH and H 2 O 2 quantification. · OH was quantified using terephthalic acid (TPA) as a hydroxyl radical dosimeter as described in previous studies 21,29 . The 3-cm-long subsamples (five per treatment) were homogenized in phosphate buffer (50 mM, pH 7.0), and the supernatant was collected after centrifugation at 10000 g for 10 min at 4 °C. The 0.2-ml extracts were incubated in a 2-ml solution containing 0.2 ml of 50 μ M TPA and 1.6 ml of phosphate buffer (50 mM, pH 7.0). After incubation for 10 min, the fluorescence emission spectra from 350 to 550 nm of monohydroxy terephthalate (TPA-· OH) was recorded with a fluorescence spectrophotometer (LS 55, Perkin-Elmer, USA) with an excitation wavelength of 326 nm.
Scientific RepoRts | 6:36276 | DOI: 10.1038/srep36276 H 2 O 2 was detected using a fluorescence spectrophotometer (LS 55, Perkin-Elmer, USA) as previously described 18,31 . The 3-cm-long subsamples (five per treatment) were homogenized in phosphate buffer (20 mM, pH 6.0). After the homogenate was centrifuged at 10000 g for 10 min at 4 °C, 5 ml of the supernatant was collected. The 3-ml reaction mixture also included 0.2 ml of root extract, 5 μ M scopoletin (S2500, Sigma), and 3 μ g ml −1 horseradish peroxidase. The fluorescence emission spectra were recorded from 400 to 550 nm with an excitation wavelength of 346 nm. Malondialdehyde (MDA) quantification. Root samples were homogenized with 0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid. The mixture was incubated at boiling water for 30 min and then quickly cooled in a refrigerator. After centrifugation at 1800 g for 10 min, the supernatant was used for MDA determination using a UV spectrophotometer (Lambda 650, Perkin-Elmer, USA) 32 .

Data analysis.
Results are shown as means ± standard deviations (SDs). One-way analyses of variance (ANOVAs) were used to determine the effects of treatment on Evans blue staining, XTT-O 2 .− formation and MDA quantification. When effects were significant, means were compared with the Tukey's test. All statistical analyses were performed by SPSS 19.0 (SPSS, Inc., USA). Differences were considered significant at P < 0.05.

Results
Aerial root viability. The surface colour of roots incubated in NH 4 NO 3 , ZnSO 4 , AlCl 3 , or FeSO 4 became darker relative to the control, while the surface colour of roots incubated in NaHSO 3 became lighter (Fig. 1). As indicated by Evans blue staining, the pollutants reduced cell viability (Fig. 1). The blue staining mainly occurred in the root tips after treatment with NaHSO 3 but occurred throughout the 3-cm-long subsample following treatment with NH 4 NO 3 , ZnSO 4 , AlCl 3 , or FeSO 4 . The absorbance of the blue extract (600 nm) confirmed that all of the pollutants significantly reduced the viability of the aerial roots (P < 0.05, Fig. 2A). Moreover, the viability was lower following NaHSO 3 , ZnSO 4 , and FeSO 4 treatment than following NH 4 NO 3 or AlCl 3 treatment.

Histochemical and cytochemical localization of O 2
.− . When dyed with NBT, root segments treated with NH 4 NO 3 , ZnSO 4 , AlCl 3 , or FeSO 4 but not with NaHSO 3 became blue, indicating the presence of O 2 .− (Fig. 1). The blue was most intense in roots treated with NH 4 NO 3 and FeSO 4 . Semi-thin transverse sections indicated that large quantities of blue formazan (NBT-O 2 .− ) accumulated in the root tips following treatment with NH 4 NO 3 (Fig. 3G-I) and the metal pollutants (Fig. 3J-R) and that most of the O 2 .− was in the root cortex and pith cells. In contrast, cross sections of control root tips or those treated with NaHSO 3 were not blue (Fig. 3A-F). In agreement

Histochemical localization and quantification of O 2 .− and · OH. As indicated by XTT-O 2 .− -formazan
absorbance at 470 nm, O 2 .− accumulation in aerial root cells was significantly higher in all of the pollutant treatments than in the control (P < 0.05, Fig. 2B). The significantly elevated O 2 .− accumulation induced by the pollutants is consistent with the NBT staining of root cross sections (Fig. 1).
To assess the accumulation of · OH, fluorescence spectra were detected by adding TPA to the root extracts. The TPA-· OH fluorescent emission curves peaked at 463 nm, and the intensities were much higher for roots treated with pollutants than for control roots (Fig. 4A). The peak of the relative fluorescent values of TPA-· OH was higher for FeSO 4 than for the other pollutants.   (Fig. 4B). Different from fluorescent assays, the histochemical staining of TMB-H 2 O 2 showed that root segments treated with NH 4 NO 3 , ZnSO 4 , AlCl 3 , or FeSO 4 was obvious, indicating the presence of H 2 O 2 . By contrary, the staining of TMB-H 2 O 2 on NaHSO 3 treated root samples was not detected (Fig. 1).
Quantification of MDA. The MDA contents of pollutant-treated aerial root samples were mostly higher than that of controls. The significantly increased MDA levels were detected in all pollutant treated root samples, indicating higher oxidative damage and lipid peroxidation (Fig. 2C).

Discussion
In this study, aerial roots of Chinese Banyan obviously suffered from treatment with pollutants as indicated by darker root surfaces (except in the case of NaHSO 3 ), dehydration symptoms (especially in the case of FeSO 4 ), and accumulation of ROS. Bisulfite (HSO 3 − ) is the byproduct of SO 2 in cells, and the derivative is directly and indirectly toxic to plant tissues 2 . SO 2 and its derivate HSO 3 − harm leaves by generating excessive quantities of ROS, resulting in the bleaching of photosynthetic pigments 33,34 . Our study found, for the first time to our knowledge, that aerial root systems were also harmed by bleaching caused by HSO 3 − , the cell death caused by NaHSO 3 was confirmed by Evans blue staining (Fig. 1). Similarly, Evans blue staining in this study indicated that the viability of aerial root cells was decreased by NH 4 NO 3 , ZnSO 4 , AlCl 3 , and FeSO 4 (Fig. 1). The decrease of aerial root cell viability was mainly caused by the decrease of cell pH, imbalance of mineral assimilation, as well as injuries in cell wall, plasma membrane, and signal transduction pathways [11][12][13][14][15][16] .
Under biotic and abiotic stress, plant cells produce ROS in several subcellular compartments 35 . As revealed by previous studies, redox-active metals (e.g., Fe 2+ and Zn 2+ ) as well as redox-inactive metals (e.g., Al 3+ ) may induce the activity of plasma membrane-localized NADPH oxidase, which transfers electrons from cytosolic NADPH to O 2   .− in cells, as indicated by biochemical assay (Fig. 2B) and by histochemical staining (Fig. 3). The exception was that only low levels of O 2 .− were detected in NaHSO 3 treated root segments: even though XTT-O 2 .− absorbance was high, NBT-O 2 .− -formazan was almost undetectable by histochemical and cytochemical observation (Fig. 3D-F). NaHSO 3 is usually used as an additive bleaching agent. Therefore, we suspect that the bleaching caused by HSO 3 − may result in the failure of NBT staining and that NBT-O 2 .− staining is not suitable for O 2 .− detection in SO 2 -or HSO 3 − -treated tissues. In our study, H 2 O 2 accumulation was not detected in pollutant-treated tissues by the fluorometric scopoletin oxidation assay (Fig. 4B). The histochemical staining, however, clearly indicated the production of H 2 O 2 in pollutant-treated aerial root samples. Here, we infer that H 2 O 2 detective method by fluorescence intensity of H 2 O 2 -formazan may not always be effective, because the peroxide activity might be enhanced during the preparation of root extract, causing more consumption of H 2 O 2 and reduced fluorescence intensity of H 2 O 2 -fomazan. This result was also in agree with previous study that H 2 O 2 is a versatile member of ROS network and that H 2 O 2 increased in plant tissues under Al stress 38-40 . · OH is among the most toxic of the ROS because of its capacity to initiate radical chain reactions that result in irreversible chemical modifications of various cellular components 41 . Because different pollutants (SO 2 , NH 4 NO 3 , and metal ions) are all involved in the accumulation of ROS within plant cells 2,20,29 , the induced oxidative processes finally break the free radical chains of membrane lipids, causing membrane decomposition (increased MDA content, Fig. 2C) and cell death (decreased cell viability, Fig. 2A). In our study, TPA-· OH fluorescence was greatly increased by all five pollutants, indicating that these pollutants increased · OH accumulation in aerial root tissues (Fig. 4A). Because Fe 2+ is involved in the Fenton reaction, FeSO 4 treatment greatly increased · OH concentrations in aerial root tissues. NH 4 NO 3 treatment also greatly increased · OH accumulation in aerial root tissue, which is consistent with previous findings that nitrate assimilation directly interferes with free radical metabolism and causes free radical-induced injury 20 .
Overall, the pollutant treatments in the current study caused ROS accumulation and profound oxidative damage, and finally cell death in aerial root tissues. Because O 2 .− is the initial ROS generated during O 2 metabolism in plant tissue, quantification of O 2 .− is vital for assessing ROS damage in plant tissues subjected to various stresses. In our study, we used both XTT and NBT to detect the accumulation of O 2 .− . XTT is more sensitive than NBT, and XTT-O 2 .− can be quantitatively detected using spectrochemical methods. NBT staining may be suitable for the qualitative assessment of O 2 .− accumulation in plant tissues that have been subjected to most stresses but not to NaHSO 3 . The bleaching effect of HSO 3 − reduced the effectiveness of NBT staining in plant tissues. This study also showed that the aerial roots of Ficus microcarpa are sensitive to various pollutants and that aerial roots may be good indicators of pollutants in industrially polluted regions.