Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

Hazardous effect of organophosphate compound, dichlorvos in transgenic Drosophila melanogaster (hsp70-lacZ): induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase

We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO(4) (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and 1.0 ppb dichlorvos as Low Observed Adverse Effect Level (LOAEL).     

Induction of biochemical stress markers and apoptosis in transgenic Drosophila melanogaster against complex chemical mixtures: role of reactive oxygen species

The study was aimed to investigate the effect of leachates of solid waste from a flashlight battery factory and a pigment plant on 70 kDa heat shock protein (Hsp70) expression, generation of reactive oxygen species (ROS), antioxidant enzymes activities and apoptosis in Drosophila. Third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) were fed on diet mixed with leachates of solid wastes (0.05-2.0%, v/v) released from two industrial plants at three different pHs (7.00, 4.93 and 2.88) for 2-48 h. A concentration- and time-dependent significant change in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content was observed in the exposed larvae preceding the antioxidant enzymes activities. Mitochondria-mediated, caspase-dependent apoptotic cell death in the larvae exposed to 1.0 and 2.0% leachates of flashlight battery factory was concurrent with a significant regression in Hsp70 expression and a higher ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression in these groups indicated the important role of ROS in the leachate-induced cellular damage. Hsp70 along with antioxidant enzymes offered protection to the organisms exposed to all the tested concentrations of the leachates of pigment plant waste and 0.5% leachate of flashlight battery factory in a cooperative manner when ROS generation was less induced. Conversely, higher levels of ROS generation in the organisms treated with 1.0 and 2.0% leachate of flashlight battery factory after 24 and 48 h resulted in regression of Hsp70 expression in them leading to cell death. The study suggests that (1) leachates of flashlight battery factory waste more adversely affected the organisms in comparison to the leachates of pigment plant waste. (2) Hsp70 may be used as a biomarker of cellular damage in organisms exposed to leachates. (3) Cell based assays using D. melanogaster as an in vivo model may provide important mechanistic information about the adverse effect of xenobiotics.     

Hsp70 Induction and hsp70 Gene polymorphisms as Indicators of acclimatization under hyperthermic conditions

The possibility of using Hsp70 and hsp70 gene polymorphisms as markers of acclimatization was investigated. Volunteers (22) were subjected to an acclimatization regimen and blood analysed for Hsp70 (Hsp72) and hsp70 polymorphisms before and after a heat tolerance test. Physiological parameters denoting acclimatization, or not, were correlated to levels of Hsp70 and combination of hsp70 genes. Only individuals that acclimatized had decreased basal Hsp70 levels and increased ability to induce Hsp70 together with a specific hsp70 genotype combination. We propose that Hsp70 levels (basal vs. induced) with the genotype combination have the potential to be used as markers of acclimatization.     

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

How do plants cope with oxidative stress in nature? A study on the dwarf bearded iris (Iris pumila)

Oxidative stress results from incongruity between the generation of toxic reactive oxygen species (ROS) and the availability of their scavengers—antioxidants. Although the short-term effects of this phenomenon are attracting much scientific attention, oxidative stress may influence an organism’s metabolism over the long (evolutionary) time scale as well. To disentangle the impact of strong light intensity from co-occurring abiotic stresses in creating adaptive responses in antioxidants and heat shock proteins (Hsps), an environment manipulation experiment was performed using a xerophyte clonal monocot, Iris pumila, native to semi-arid grasslands at the Deliblato Sands. This species is very tolerant to the combined effect of extreme abiotic stressors such as high light intensity, elevated soil surface temperatures, and scarcity of water, which commonly takes place in its natural habitats during the summer. By shading half of each selected clone, leaving the other half sun-exposed, we contrasted short-term effects of reduced daylight intensity with long-term effects of photo-oxidative stress. In both light treatments, the enzymatic activities of SOD and APX antioxidants were similar in magnitude, whereas those of CAT and POD significantly decreased in exposed compared to shaded leaves. Moreover, exposed leaves expressed a unique CAT isoform that differed biochemically from two CAT isoforms observed in shaded leaves. The content of non-enzymatic antioxidants, carotenoids (Car), remained constant with the reduction of light intensity, but their ratio to total chlorophylls (Chl) significantly decreased compared to that expressed in full sunlight. The abundance of Hsps was considerably greater in exposed than in shaded leaves, especially regarding the inducible isoforms, Hsp70 and Hsp90a, as were their proportions in relation to the constitutively expressed Hsp90b isoform. The presented results, thus, indicate that adaptive metabolic responses of I. pumila leaves to photo-oxidative stress entailed the high activity of two key enzymatic antioxidants, SOD and APX and the expression of a light-resistant CAT—to counteract the stress-mediated ROS accumulation, the increased Car to Chl ratio—to adjust the photosynthetic apparatus to the high light conditions, as well as the accelerated biosynthesis of heat shock proteins Hsp70 and Hsp90—to preserve the cellular proteostasis.     

Cellular internalization and stress response of ingested amorphous silica nanoparticles in the midgut of Drosophila melanogaster

Background

Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (< 30 nm) in the midgut of Drosophila melanogaster (Oregon R⁺) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles.

Methods

Third instar larvae of D. melanogaster were exposed orally to 1–100 μg/mL of aSNPs for 12–36 h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints.

Results

A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration.

Conclusion

aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death.

General significance

Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

Ayurvedic <Emphasis Type="Italic">Amalaki Rasayana</Emphasis> promotes improved stress tolerance and thus has anti-aging effects in <Emphasis Type="BoldItalic">Drosophila melanogaster</Emphasis>

Amalaki Rasayana (AR) is a common Ayurvedic herbal formulation of Phyllanthus emblica fruits and some other ingredients, and is used for general good health and healthy aging. We reported it to improve life history traits and to suppress neurodegeneration as well as induced apoptosis in Drosophila. The present study examines responses of Drosophila reared on AR-supplemented food to crowding, thermal or oxidative stresses. Wild-type larvae/flies reared on AR-supplemented food survived the various cell stresses much better than those reared on control food. AR-fed mutant park ¹³ or DJ-1β ^Delta93 (Parkinson’s disease model) larvae/flies, however, showed only partial or no protection, respectively, against paraquat-induced oxidative stress, indicating essentiality of DJ-1β for AR-mediated oxidative stress tolerance. AR feeding reduced the accumulation of reactive oxygen species (ROS) and lipid peroxidation even in aged (35-day-old) wild-type flies while enhancing superoxide dismutase (SOD) activity. We show that while Hsp70 or Hsp83 expression under normal or stress conditions was not affected by AR feeding, Hsp27 levels were elevated in AR-fed wild-type control as well as heat-shocked larvae. Therefore, besides the known anti-oxidant activity of Phyllanthus emblica fruits, dietary AR also enhances cellular levels of Hsp27. Our in vivo study on a model organism shows that AR feeding significantly improves tolerance to a variety of cell stresses through reduced ROS and lipid peroxidation on the one hand, and enhanced SOD activity and Hsp27 on the other. The resulting better homeostasis improves life span and quality of organism’s life.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress

Relatively low or high temperatures are responsible for a variety of physiological stress responses in insects and mites. Induced thermal stress was recently associated with increased reactive oxygen species (ROS) generation, which caused oxidative damage. In this study, we examined the time-related effect of the relatively low (0, 5, 10, and 15 °C) or high (32, 35, 38, and 41 °C) temperatures on the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), and glutathione-S-transferase (GST), and the total antioxidant capacity (TEAC) of the citrus red mite, Panonychus citri (McGregor). The malondialdehyde (MDA) concentration, as a marker of lipid peroxidation in organisms, was also measured in the citrus red mite under thermal stress conditions. Results showed that SOD and GST activities were significantly increased and play an important role in the process of antioxidant response to thermal stress. Lipid peroxidation levels increased significantly (P < 0.001) and changed in a time-dependent manner. CAT and POX activity, as well as TEAC, did not vary significantly and play a minor role to remove the ROS generation. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play an important role in reducing oxidative damage in the citrus red mite.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Characterization and expression of cytoplasmic copper/zinc superoxide dismutase (CuZn SOD) gene under temperature and hydrogen peroxide (H2O2) in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692 bp and had a 465 bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37 °C) revealed that its expression was enhanced 4.2-fold (p < 0.001) at 30 °C after 2 h, however, the lower temperature (15 °C) promoted CuZn SOD transiently (4.1-fold, p < 0.001) and then the expression of CuZn SOD decreased to normal level (p > 0.05). When exposed to H₂O₂ (0.1 mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.     

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis

Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type “wt” strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.     

Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed

Mussels Perna perna were exposed to air for 24 h showing a clear increase in the levels of lipid peroxidation and oxidative DNA damage, measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The levels of lipid peroxidation increased both in the digestive gland and gills, while oxidative DNA damage increased only in the gills. After the 24 h of air exposure, mussels were re-submersed for a period of 3 h, leading values to return to a pre-aerial exposure levels. Control animals were kept immersed during the whole period. Several antioxidant and complementary enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and the levels of total glutathione (Total GSH) were assayed in a second set of experiments where one group of mussels were exposed to air for 18 h and other to 1 h re-submersion after 18 h aerial exposure. Only a 52% increase in the glutathione S-transferase activity was observed in the digestive gland, which remained elevated to about 40% after 1 h re-submersion, showing that defense systems can be modulated even during oxygen deprivation in P. perna. The DNA and lipid oxidative damage observed after aerial exposure indicates that mussels face an oxidative challenge, and are able to counteract such an “insult” as values of lipid peroxidation and DNA damage returned to control values after 3 h re-submersion.     

扑草净对远志幼苗根系活力及氧化胁迫的影响

以远志(Polygala tenuifolia Willd.)为材料,应用组织化学和生物化学的方法研究不同浓度扑草净(0—400 mg/L)对远志幼苗生长、根系活力、膜脂过氧化、活性氧含量及抗氧化酶活性等的影响。10 mg/L扑草净对远志幼苗根系活力、细胞膜完整性及活性氧的积累几乎无显著影响,而25—400 mg/L扑草净处理则显著增加活性氧的积累,明显抑制根系活力且破坏细胞膜完整性;上述结果进一步被膜脂过氧化、质膜完整性、活性氧产生(O.2-和H2O2)的非损伤组织化学染色所证明。远志幼苗可通过多种抗氧化酶(SOD、POD、CAT、APX等)和非酶抗氧化剂(如脯氨酸)的相互协调作用,清除低浓度扑草净胁迫诱发产生的活性氧,减轻对细胞的伤害。研究结果表明,发芽期是远志对扑草净处理的敏感时期,较为安全的扑草净临界浓度为10 mg/L;25mg/L扑草净处理即引起远志幼苗氧化胁迫和膜脂过氧化,使细胞膜的完整性受到破坏,根系活力下降,抑制了远志幼苗的生长发育。该研究为远志抗除草剂胁迫机制及其栽培过程中除草剂的安全合理使用提供理论依据。