Titanium Dioxide Nanoparticles Promote Root Growth by Interfering with Auxin Pathways in <i>Arabidopsis thaliana</i>

TiO₂ nanoparticles (nano-TiO₂) are widely used in the world, and a considerable amount of nano-TiO₂ is released into the environment, with toxic effects on organisms. In the various species of higher plants, growth, including seed germination, root elongation, and biomass accumulation, is affected by nano-TiO₂. However, the underlying molecular mechanisms remain to be elucidated. In this study, we observed that nano-TiO₂ promoted root elongation in a dose-dependent manner. Furthermore, we found that nano-TiO₂ elevated auxin accumulation in the root tips of the auxin marker lines DII-VENUS and DR5:: GUS, and, correspondingly, quantitative real-time PCR analysis revealed that nano-TiO₂ increased the expression levels of auxin biosynthesis- and transportrelated genes. GFP fluorescence observation using transgenic PIN2-GFP indicated that nano-TiO₂ promoted root growth by inducing PIN2 accumulation. Thus, we propose that nano-TiO₂ promote root growth in Arabidopsis thaliana by altering the expression levels of auxin biosynthesis- and transport-related genes.     

Nano-TiO2 Improve the Photosynthesis of Tomato Leaves under Mild Heat Stress

Nano-TiO₂ has been reported to promote photosynthesis in some crops; however, the mechanism behind this action remains unknown. In this research, the effects of nano-TiO₂ on leaf photosynthesis under mild heat stress were investigated. Results showed that the net photosynthetic rate, conductance to H₂O, and transpiration rate of tomato leaves increased after application of an appropriate concentration of nano-TiO₂. Nano-TiO₂ also significantly decreased the minimum chlorophyll fluorescence and relative electron transport in leaves. Under mild heat stress, Nano-TiO₂ increased regulated photosystem II (PS II) energy dissipation and decreased non-regulated PS II energy dissipation. These results indicate that nano-TiO₂ plays a positive role in promoting photosynthesis in tomato leaves under mild heat stress.     

Nano-TiO2 Is Not Phytotoxic As Revealed by the Oilseed Rape Growth and Photosynthetic Apparatus Ultra-Structural Response

Recently nano-materials are widely used but they have shown contrasting effects on human and plant life. Keeping in view the contrasting results, the present study has evaluated plant growth response, antioxidant system activity and photosynthetic apparatus physiological and ultrastructural changes in Brassica napus L. plants grown under a wide range (0, 500, 2500, 4000 mg/l) of nano-TiO₂ in a pot experiment. Nano-TiO₂ has significantly improved the morphological and physiological indices of oilseed rape plants under our experimental conditions. All the parameters i-e morphological (root length, plant height, fresh biomass), physiological (photosynthetic gas exchange, chlorophyll content, nitrate reductase activity) and antioxidant system (Superoxide dismutase, SOD; Guaiacol peroxidase, POD; Catalase, CAT) recorded have shown improvement in their performance by following nano-TiO₂ dose-dependent manner. No significant chloroplast ultra-structural changes were observed. Transmission electron microscopic images have shown that intact & typical grana and stroma thylakoid membranes were in the chloroplast, which suggest that nano-TiO₂ has not induced the stressful environment within chloroplast. Finally, it is suggested that, nano-TiO₂ have growth promoting effect on oilseed rape plants.     

Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytes

We investigated the signaling pathways underlying nano-TiO₂-induced apoptosis in cultured human lymphocytes. Nano-TiO₂ increased the proportion of sub-G1 cells, activated caspase-9 and caspase-3, and induced caspase-3-mediated PARP cleavage. Nano-TiO₂ also induced loss of mitochondrial membrane potential, which suggests that nano-TiO₂ induces apoptosis via a mitochondrial pathway. A time-sequence analysis of the induction of apoptosis by nano-TiO₂ revealed that nano-TiO₂ triggered apoptosis through caspase-8/Bid activation. We also observed that inhibition of caspase-8 by z-IETD-fmk suppressed the caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and apoptosis. Nano-TiO₂ activated two MAPKs, p38 and JNK. In addition, the selective p38 inhibitor SB203580 and selective JNK inhibitor SP600125 suppressed nano-TiO₂-induced apoptosis and caspase-8 activation to moderate and significant extents, respectively. Knockdown of protein levels of JNK1 and p38 using an RNA interference technique also suppressed caspase-8 activation. Our results suggest that nano-TiO₂-induced apoptosis is mediated by the p38/JNK pathway and the caspase-8-dependent Bid pathway in human lymphocytes.     

Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach

The effects of nano-TiO₂ (rutile) and non-nano-TiO₂ on the germination and growth of naturally aged spinach seeds were studied by measuring the germination rate and the germination and vigor indexes of aged spinach seeds. An increase of these factors was observed at 0.25–4‰ nano-TiO₂ treatment. During the growth stage, the plant dry weight was increased, as was the chlorophyll formation, the ribulosebisphosphate carboxylase/oxygenase activity, and the photosynthetic rate. The best results were found at 2.5‰ nano-TiO₂. The effects of non-nano-TiO₂ are not significant. It is shown that the physiological effects are related to the nanometer-size particles, but the mechanism by which nano-TiO₂ improves the growth of spinach seeds still needs further study.     

Molecular Mechanisms of Nanosized Titanium Dioxide–Induced Pulmonary Injury in Mice

The pulmonary damage induced by nanosized titanium dioxide (nano-TiO₂) is of great concern, but the mechanism of how this damage may be incurred has yet to be elucidated. Here, we examined how multiple genes may be affected by nano-TiO₂ exposure to contribute to the observed damage. The results suggest that long-term exposure to nano-TiO₂ led to significant increases in inflammatory cells, and levels of lactate dehydrogenase, alkaline phosphate, and total protein, and promoted production of reactive oxygen species and peroxidation of lipid, protein and DNA in mouse lung tissue. We also observed nano-TiO₂ deposition in lung tissue via light and confocal Raman microscopy, which in turn led to severe pulmonary inflammation and pneumonocytic apoptosis in mice. Specifically, microarray analysis showed significant alterations in the expression of 847 genes in the nano-TiO₂-exposed lung tissues. Of 521 genes with known functions, 361 were up-regulated and 160 down-regulated, which were associated with the immune/inflammatory responses, apoptosis, oxidative stress, the cell cycle, stress responses, cell proliferation, the cytoskeleton, signal transduction, and metabolic processes. Therefore, the application of nano-TiO₂ should be carried out cautiously, especially in humans.     

Functional diversity in fruit‐frugivore interactions: a field experiment with Mediterranean mammals

Using field seed sowings, we assessed how four mammal species (Meles meles, Vulpes vulpes, Sus scrofa, and Oryctolagus cuniculus) influenced seed germination in three fleshy‐fruited Mediterranean shrubs (Corema album, Pyrus bourgaeana, and Rubus ulmifolius). We predicted that gut passage and removal away from mother plants would enhance the quantity, speed, and asynchrony of seed germination. Results showed that percent germination was altered by gut passage, but that the magnitude and even the direction of such effects varied according to plant and disperser species. Likewise, dispersal away from mother plants affected the percentage and germination speed in some species but not others. Gut passage increased asynchrony of germination in Rubus and Pyrus, and removal from the mother plant increased asynchrony in Rubus, which likely enhances plant fitness in unpredictable environments. Gut passage generally had a stronger effect on germination than removal away from mother plants, but for some species both factors were similarly influential. Therefore, the combined effects of both seed dispersal services varied individually among fruit and frugivore species, leading to unusually high functional diversity in this seed dispersal mutualism.     

Effects of Aflatoxin on Seeding Growth and Ultrastructure in Plants

Nineteen plants belonging to 11 species of the cruciferae were studied to determine the effects of aflatoxin B₁ on seed germination and seedling development. Germination was not inhibited in any test organism at a concentration of 100 μg of aflatoxin per ml of agar substrate. Inhibition of elongation of the hypocotyls and roots in the species studied varied from 29 to 93% and from 22 to 91% in the respective tissues. Lepidium sativum was the most susceptible plant studied and exhibited the maximal inhibitory response noted above at concentrations of 8 μg of aflatoxin per ml. The ultrastructure of Lepidium root cells treated with crystalline aflatoxin B₁ exhibited morphological changes characteristic of those found in aflatoxin-treated animal cells. In addition to changes in the cytoplasmic organelles, numerous ring-shaped nucleoli with prominent nucleolar caps were produced. The effect of aflatoxin on plant cells is compared with similar effects induced by actinomycin D. Seed germination and seedling development is discussed in relation to the effects of both compounds on deoxyribonucleic acid-dependent ribonucleic acid biosynthesis.     

Growth response of komatsuna (Brassica rapa var. peruviridis) to root and foliar applications of phosphite

Soil and hydroponic culture experiments were conducted to investigate the effects of phosphite (Phi) as phosphorus (P) fertilizer via root and foliar applications on the growth and P supply of komatsuna. In both experiments, root P treatments were combinations of Phi and phosphate (Pi) at different Pi:Phi ratios, for a total of high P level (92 mg P pot^?1; the soil experiment) or low P level (0.05 mM P; the hydroponic experiment). Foliar P treatments were deionized water (control), a Pi solution and a Phi solution at low concentration of 0.05% P₂O₅. In both experiments, shoot dry weight of plants significantly decreased as Pi:Phi ratio decreased. In the soil experiment, plants grew abnormally at a Pi:Phi ratio of 25:75 and died when P was applied to soil entirely as Phi form (0:100 treatment). In the hydroponic experiment, no visible damage was found in shoot but root growth was strongly inhibited with severe damage symptoms at low Pi:Phi ratios. Total P concentration in plant decreased significantly with decreasing Pi:Phi ratio, especially in the hydroponic experiment. Foliar application of Phi although greatly increased total P of plants compared to that of Pi in both experiments, it did not improve but further decreased plant growth at low Pi:Phi ratios in the soil experiment and at all Pi:Phi ratios in the hydroponic experiment. The results of this study clearly indicated that Phi could not be used as P fertilizer by komatsuna plants via both application methods and could not substitute P at any rate at either low or high level. No beneficial effect of Phi was detected even when it was applied at low rate or applied in combination with Pi at different ratios. The effects of Phi were strongly dependent on the P nutrition status of plants; and plants that were not sufficiently fertilized with Pi may become vulnerable to Phi even at low levels.     

Nanosized TiO2-Induced Reproductive System Dysfunction and Its Mechanism in Female Mice

Recent studies have demonstrated nanosized titanium dioxide (nano-TiO₂)-induced fertility reduction and ovary injury in animals. To better understand how nano-TiO₂ act in mice, female mice were exposed to 2.5, 5, and 10 mg/kg nano-TiO₂ by intragastric administration for 90 consecutive days; the ovary injuries, fertility, hormone levels, and inflammation-related or follicular atresia-related cytokine expression were investigated. The results showed that nano-TiO₂ was deposited in the ovary, resulting in significant reduction of body weight, relative weight of ovary and fertility, alterations of hematological and serum parameters and sex hormone levels, atretic follicle increases, inflammation, and necrosis. Furthermore, nano-TiO₂ exposure resulted in marked increases of insulin-like growth factor-binding protein 2, epidermal growth factor, tumor necrosis factor-α, tissue plasminogen activator, interleukin-1β, interleukin -6, Fas, and FasL expression, and significant decreases of insulin-like growth factor-1, luteinizing hormone receptor, inhibin α, and growth differentiation factor 9 expression in mouse ovary. These findings implied that fertility reduction and ovary injury of mice following exposure to nano-TiO₂ may be associated with alteration of inflammation-related or follicular atresia-related cytokine expressions, and humans should take great caution when handling nano-TiO₂.     

Reactive oxygen species,ABA and nitric oxide interactions on the germination of warm-season C<Subscript>4</Subscript>-grasses

Hydrogen peroxide (H₂O₂) as a source of reactive oxygen species (ROS) significantly stimulated germination of switchgrass (Panicum virgatum L.) seeds with an optimal concentration of 20 mM at both 25 and 35°C. For non-dormant switchgrass seeds exhibiting different levels of germination, treatment with H₂O₂ resulted in rapid germination (<3 days) of all germinable seeds as compared to seeds placed on water. Exposure to 20 mM H₂O₂ elicited simultaneous growth of the root and shoot system, resulting in more uniform seedling development. Seeds of big bluestem (Andropogon gerardii Vitman) and indiangrass [Sorghastrum nutans (L.) Nash] also responded positively to H₂O₂ treatment, indicating the universality of the effect of H₂O₂ on seed germination in warm-season prairie grasses. For switchgrass seeds, abscisic acid (ABA) and the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI) at 20 μM retarded germination (radicle emergence), stunted root growth and partially inhibited NADPH-oxidase activity in seeds. H₂O₂ reversed the inhibitory effects of DPI and ABA on germination and coleoptile elongation, but did not overcome DPI inhibition of root elongation. Treatment with H₂O₂ appeared to enhance endogenous production of nitric oxide, and a scavenger of nitric oxide abolished the peroxide-responsive stimulation of switchgrass seed germination. The activities and levels of several proteins changed earlier in seeds imbibed on H₂O₂ as compared to seeds maintained on water or on ABA. These data demonstrate that seed germination of warm-season grasses is significantly responsive to oxidative conditions and highlights the complex interplay between seed redox status, ABA, ROS and NO in this system.     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Benzoxazolin-2-(3<Emphasis Type="Italic">H</Emphasis>)-one reduces photosynthetic activity and chlorophyll fluorescence in soybean

Benzoxazolin-2-(3H)-one (BOA) has been tested in many plants species, but not in soybean (Glycine max). Thus, a hydroponic experiment was conducted to assess the effects of BOA on soybean photosynthesis. BOA reduced net photosynthetic rate, stomatal conductance, and effective quantum yield of PSII photochemistry without affecting intercellular CO₂ concentration or maximal quantum yield of PSII photochemistry. Results revealed that the reduced stomatal conductance restricted entry of CO₂ into substomatal spaces, thus limiting CO₂ assimilation. No change found in intercellular CO₂ concentration and reduced effective quantum yield of PSII photochemistry revealed that CO₂ was not efficiently consumed by the plants. Our data indicated that the effects of BOA on soybean photosynthesis occurred due to the reduced stomatal conductance and decreased efficiency of carbon assimilation. The accumulation of BOA in soybean leaves reinforced these findings.     

Acute and chronic ozone exposure temporarily affects seed germination in alpine plants

This study was the first to investigate the direct effects of anomalous concentrations of ozone mediated by summer heat waves on seed germination in alpine plants. During germination, the seeds were exposed to three peaks of O₃ concentration (125 ppb for 5 and 10 days; 185 ppb for 5 days), derived from measurements taken close to the species growing site. High O₃ concentration delayed the first germination time, increased the mean germination time, and reduced the germination percentage during and immediately after the treatment, but, in most cases, effects were weak and had almost vanished three weeks after the treatments. In few cases, chronic exposure to O₃ (125 for 10 days’ treatment) enhanced seed germination compared to the control, suggesting that ozone may induce antioxidant and DNA-repair mechanisms or dormancy-breaking effects in hydrated seeds. Although seed mortality increased during O₃ treatments in four species, the effect of O₃ on seed germination is mostly limited to the period of exposure, indicating that it is unlikely to produce permanent negative effects on seeds, during the germination phase. Our results show that the direct effect of O₃ on seeds of alpine plants may have minor impacts on plant reproductive performance during seed germination.     

Environmental control of growth and development of Scrophularia marilandica

Summary Seedlings of Scrophularia marilandica were grown at different combinations of day/night temperature and photoperiod under controlled conditions. The species flowered in long days. The stems of plants grown at low temperature and short photoperiod failed to elongate. Treatment with gibberellic acid (GA₃) simulated the effect of increasing temperature and photoperiod and caused stem elongation in plants which would otherwise not have elongated. Application of GA₃ to plants grown at high temperature and long photoperiod resulted in increased stem elongation and flowering. The growth retardant (2-chloroethyl)trimethylammonium chloride (CCC) had little effect on rosette plants grown at low temperature and short photoperiod. Application of CCC to +GA₃ plants grown at a higher temperature and long photoperiod gave a significant increase in stem height. The interaction between temperature and applied GA is described in an experiment using plants grown at high and low temperatures for varying periods of time.This work was supported by National Science Foundation Grant GB 17483.     

Hydrogen peroxide effects on root hydraulic properties and plasma membrane aquaporin regulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

In the last few years, the role of reactive oxygen species as signaling molecules has emerged, and not only as damage-related roles. Here, we analyzed how root hydraulic properties were modified by different hydrogen peroxide (H₂O₂) concentrations applied exogenously to the root medium. Two different experimental setups were employed: Phaseolus vulgaris plants growing in hydroponic or in potted soils. In both experimental setups, we found an increase of root hydraulic conductance (L) in response to H₂O₂ application for the first time. Twenty millimolar was the threshold concentration of H₂O₂ for observing an effect on L in the soil experiment, while in the hydroponic experiment, a positive effect on L was observed at 0.25 mM H₂O₂. In the hydroponic experiment, a correlation between increased L and plasma membrane aquaporin amount and their root localization was observed. These findings provide new insights to study how several environmental factors modify L.     

Differential effects of camptothecin and interactions with plant hormones on seed germination and seedling growth

Effects of camptothecin, a naturally occurring alkaloid, on seed germination varied from promotive to inhibitory, depending on the species used. It markedly inhibited seedling root growth but its inhibition of hypocotyl growth varied among species. Camptothecin inhibited GA₃-induced dark germination of lettuce (Lactuca sativa L.) seeds and hypocotyl elongation of seedlings. In contrast to ABA, the camptothecin inhibition of GA₃-induced germination could not be overcome by cytokinin. When seeds were germinated at 29C with a 0.5 h light treatment, little or no germination occurred in the camptothecin treatment, but addition of cytokinin overcame this inhibition.     

ELEMENT RESPONSES OF AGAVES

To help identify possible element stresses, seedling responses of Agave deserti to high concentrations of various elements were examined by monitoring both 12-day growth in hydroponic solution and 6-month growth in sand culture. In addition, nocturnal acid accumulation by adult plants of six agave species was related to element levels in their chlorenchyma. Compared with common agronomic plants, seedlings of A. deserti were quite sensitive to salinity, with 50 mm NaCl greatly reducing root elongation in hydroponic solution and watering with 25 mm NaCl preventing growth in sand culture. The seedlings were rather insensitive to Ca concentrations from 0.2 to 5 mm and to pH from pH 5 to 8. They also tolerated high levels of B and of the heavy metals Cu and Zn. Nocturnal acid accumulation by adult plants of the six agave species was positively correlated with levels of 10 elements in the chlorenchyma, especially N (r² = 0. 70), B (r² = 0.51), and Ca (r² = 0.46). In contrast, nocturnal acid accumulation was weakly and negatively correlated with chlorenchyma Na (r² = 0.13), consistent with the deleterious effects of salinity on the growth of seedlings. Correlations between nocturnal acid accumulation and element content were consistent with previous fertilizer experiments with N, B, Ca, K, and P on A. sisalana. Element levels in the chlorenchyma of the six agave species were generally similar to those of previously studied cacti, including a low Na and high Ca level compared with agronomic plants.     

Effects of Silver Nanoparticle Exposure on Germination and Early Growth of Eleven Wetland Plants

The increasing commercial production of engineered nanoparticles (ENPs) has led to concerns over the potential adverse impacts of these ENPs on biota in natural environments. Silver nanoparticles (AgNPs) are one of the most widely used ENPs and are expected to enter natural ecosystems. Here we examined the effects of AgNPs on germination and growth of eleven species of common wetland plants. We examined plant responses to AgNP exposure in simple pure culture experiments (direct exposure) and for seeds planted in homogenized field soils in a greenhouse experiment (soil exposure). We compared the effects of two AgNPs–20-nm polyvinylpyrrolidine-coated silver nanoparticles (PVP-AgNPs) and 6-nm gum arabic coated silver nanoparticles (GA-AgNPs)–to the effects of AgNO₃ exposure added at equivalent Ag concentrations (1, 10 or 40 mg Ag L⁻¹). In the direct exposure experiments, PVP-AgNP had no effect on germination while 40 mg Ag L⁻¹ GA-AgNP exposure significantly reduced the germination rate of three species and enhanced the germination rate of one species. In contrast, 40 mg Ag L⁻¹ AgNO₃ enhanced the germination rate of five species. In general root growth was much more affected by Ag exposure than was leaf growth. The magnitude of inhibition was always greater for GA-AgNPs than for AgNO₃ and PVP-AgNPs. In the soil exposure experiment, germination effects were less pronounced. The plant growth response differed by taxa with Lolium multiflorum growing more rapidly under both AgNO₃ and GA-AgNP exposures and all other taxa having significantly reduced growth under GA-AgNP exposure. AgNO₃ did not reduce the growth of any species while PVP-AgNPs significantly inhibited the growth of only one species. Our findings suggest important new avenues of research for understanding the fate and transport of NPs in natural media, the interactions between NPs and plants, and indirect and direct effects of NPs in mixed plant communities.     

Nano-TiO<Subscript>2</Subscript> induces autophagy to protect against cell death through antioxidative mechanism in podocytes

Autophagy is a cellular pathway involved in degradation of damaged organelles and proteins in order to keep cellular homeostasis. It plays vital role in podocytes. Titanium dioxide nanoparticles (nano-TiO₂) are known to induce autophagy in cells, but little has been reported about the mechanism of this process in podocytes and the role of autophagy in podocyte death. In the present study, we examined how nano-TiO₂ induced authophagy. Besides that, whether autophagy could protect podocytes from the damage induced by nano-TiO₂ and its mechanism was also investigated. Western blot assay and acridine orange staining presented that nano-TiO₂ significantly enhanced autophagy flux in podocytes. In addition, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were involved in such process. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that upregulated level of autophagy induced by rapamycin in high concentration nano-TiO₂-treated podocytes could significantly reduce the level of oxidative stress and alleviate podocyte death. Downregulating the level of autophagy with 3-methyladenine had the opposite effects. These findings indicate that nano-TiO₂ induces autophagy through activating AMPK to inhibit mTOR in podocytes, and such autophagy plays a protecting role against oxidative stress on the cell proliferation. Changing autophagy level may become a new treatment strategy to relieve the damage induced by nano-TiO₂ in podocytes.