Improvement of element uptake and antioxidative defense in Brassica napus under lead stress by application of hydrogen sulfide

Heavy metal pollution is one of the major constraints in oilseed rape (Brassica napus L.) production. In this study, protective role of hydrogen sulfide (H₂S) on plant growth under lead (Pb) stress was studied in B. napus. Plants were grown hydroponically in greenhouse conditions under three levels (0, 100, and 400 μM) of Pb and three levels (0, 100 and 200 μM) of H₂S donor sodium hydrosulfide. Outcomes demonstrated that Pb stress significantly reduced the plant biomass, leaf chlorophyll contents, nutrients uptake in the leaves and roots of B. napus plants. Exogenous application of H₂S significantly improved the plant biomass, chlorophyll contents and concentration of macro- and micronutrients in the leaves and roots of B. napus plants under Pb-toxicity conditions. The data indicated that application of Pb alone significantly increased the reactive oxygen species (ROS) as well as malondialdehyde (MDA) in the leaves and roots of plants. Meanwhile, application of H₂S decreased the production of MDA and ROS in the leaves and roots by increasing antioxidant activities under Pb stress. Moreover, this study also revealed that plants treated with H₂S at different concentrations enhanced the contents of total glutathione and glutathione reduced/glutathione oxidized ratio in leaves and roots under different levels of Pb. The results depicted that H₂S improved the plant biomass, uptake of nutrients in the leaves and roots of B. napus plants and enhanced the performance of antioxidant defense system due to its ameliorative potential under Pb stress conditions.     

The ecotoxicological and interactive effects of chromium and aluminum on growth,oxidative damage and antioxidant enzymes on two barley genotypes differing in Al tolerance

The effects of single or combined stress of aluminum (Al) and chromium (Cr) on plant growth, root dehydrogenase, oxidative stress and antioxidative enzymes were studied using two barley genotypes differing in Al tolerance in a hydroponic experiment. Al or Cr stress decreased plant growth, lowered root dehydrogenase activity and caused oxidative damage, as characterized by increased MDA and H₂O₂ contents. Under Al or Cr stress, the activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), were dramatically increased in plant tissues. Gebeina, an Al-tolerant genotype, had less oxidative damage than Shang 70-119, an Al-sensitive genotype. The extent of oxidative damage induced by Cr varied with the pH of the culture solution, with lower pH values (4.0) being more severe than higher pH values (6.5). The combination of Cr and Al caused a further decrease in plant growth, a decrease in root dehydrogenase activity and an increase in MDA and H₂O₂ contents as well as the activities of antioxidative enzymes. There was also a marked difference between the two barley genotypes in the extent of increased antioxidative enzyme activity under the Cr and Al stresses.     

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H₂O₂ generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H₂O₂. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.     

Alleviation of osmotic stress in Brassica napus,B. campestris,and B. juncea by ascorbic acid application

The roles of ascorbic acid (AsA, 1 mM) under an osmotic stress [induced by 15 % (m/v) polyethylene glycol, PEG-6000] were investigated by examining morphological and physiological attributes in Brassica species. The osmotic stress reduced the fresh and dry masses, leaf relative water content (RWC), and chlorophyll (Chl) content, whereas increased the proline (Pro), malondialdehyde (MDA), and H₂O₂ content, and lipoxygenase (LOX) activity. The ascorbate content in B. napus, B. campestris, and B. juncea decreased, increased, and remained unaltered, respectively. The dehydroascorbate (DHA) content increased only in B. napus. The AsA/DHA ratio was reduced by the osmotic stress in all the species except B. juncea. The osmotic stress increased the glutathione (GSH) content only in B. juncea, but increased the glutathione disulfide (GSSG) content and decreased the GSH/GSSG ratio in all the species. The osmotic stress increased the activities of ascorbate peroxidase (APX) (except in B. napus), glutathione reductase (GR) (except in B. napus), glutathione S-transferase (GST) (except in B. juncea), and glutathione peroxidase (GPX), and decreased the activities of catalase (CAT) and monodehydroascorbate reductase (MDHAR) (only in B. campestris). The osmotic stress decreased the glyoxalase I (Gly I) and increased glyoxalase II (Gly II) activities. The application of AsA in combination with PEG improved the fresh mass, RWC, and Chl content, whereas decreased the Pro, MDA, and H₂O₂ content in comparison with PEG alone. The AsA addition improved AsA-GSH cycle components and improved the activities of all antioxidant and glyoxalase enzymes in most of the cases. So, exogenous AsA improved physiological adaptation and alleviated oxidative damage under the osmotic stress by improving the antioxidant and glyoxalase systems. According to measured parameters, B. juncea can be recognized as more drought tolerant than B. napus and B. campestris.     

Effect of Aluminium on Oxidative Stress Related Enzymes Activities in Barley Roots

The impact of aluminium stress on activities of enzymes of the oxidative metabolism: superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), NADH peroxidase (NADH-POD) and oxalate oxidase (OXO) was studied in barley (Hordeum vulgare L. cv. Alfor) root tips. SOD appeared to be involved in detoxification mechanisms at highly toxic Al doses and after long Al exposure. POD and APX, H₂O₂ consuming enzymes, were activated following similar patterns of expression and exhibiting significant correlation between their elevated activities and root growth inhibition. The signalling role of NADH-POD in oxidative stress seems to be more probable than that of OXO, which might be involved in Al toxicity mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.     

外源硫化氢对豌豆根尖及其边缘细胞的影响

为研究外源硫化氢(H2S)对豌豆(Pisum sativum)胚根生理特性及其边缘细胞的影响,测定了不同浓度外源H2S处理下豌豆根长、根尖组织可溶性蛋白质含量、抗氧化酶(APX、CAT、POD和SOD)活性、根尖边缘细胞存活率及其粘胶层相对面积的变化。结果表明:低浓度(0-40μmol·L-1)H2S可促进豌豆胚根生长,根尖组织可溶性蛋白质含量升高,SOD、APX和POD活性增加,CAT活性降低,根尖边缘细胞存活率上升,粘胶层相对面积变小。高浓度(60-80μmol·L-1)H2S可抑制豌豆胚根生长,可溶性蛋白质含量和边缘细胞存活率明显下降,APX和POD活性降低,CAT活性升高,SOD活性没有明显变化,粘胶层相对面积变大;边缘细胞染色体凝集并边缘化,然后逐渐降解并伴随粉末化,细胞质膜皱缩。因此,推测H2S可能在植物体内发挥着重要的生理作用。     

Selenium Inhibits Root Elongation by Repressing the Generation of Endogenous Hydrogen Sulfide in Brassica rapa

Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H₂S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H₂S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H₂S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H₂S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H₂S by the application of H₂S donor NaHS could significantly alleviate Se(IV)-induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H₂S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H₂S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H₂S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H₂S in modulating Se(IV)-induced phytotoxicity in roots.     

Silicon supplementation improves drought tolerance in canola plants

This paper reports the effects of silicon (Si) supplementation (0.35 g Na₂SiO₃/kg soil, 2.73 mmol Si/kg soil) on some physiological characteristics of canola (Brassica napus L. cv. Okapi) plants grown in pots and treated with or without Si under water stress for 25 days. In this study, Si-supplied plants showed the higher shoot dry weight (SDW), root dry weight (RDW), relative growth rate (RGR), net assimilation rate (NAR), and relative water content (RWC) as compared to those without application of Si under drought conditions. However, Si did not significantly affect dry weight accumulation under well-watered conditions. CO₂ absorbance (A) was increased by silicon under water stress conditions. The intracellular CO₂ concentration (C _i), which was significantly elevated under water stress, was decreased by Si. Silicon application significantly increased the root amino acid content after 20 and 25 days of water stress. In Si-supplemented water-deficit plants, the amount of MDA remained unchanged after 25 days of water stress, obviously because of an efficient scavenging following significant enhancement of superoxide dismutase (SOD) and peroxidase (POD) activities. These results indicated that Si ameliorated growth reduction of drought-stressed canola plants, accompanied by an increase in the root water uptake through the formation of more fine roots and more amino acids as osmotica for supporting water uptake. The results of this study revealed that Si application alleviated water stress damages because of the higher water uptake, better photosynthetic rate, and the lowered lipid peroxidation in canola plants.     

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

Aims

Aluminum (Al) toxicity is one of the major factors that limit plant growth. Low concentration of hydrogen sulfide (H₂S) has been proven to function in physiological responses to various stresses. The objective of this study is to investigate the possible role of H₂S in Al toxicity in barley (Hordeum vulgare L) seedlings.

Methods

Barley seedlings pre-treated with sodium hydrosulfide (NaHS), a H₂S donor, and subsequently exposed to Al treatment were studied for their effects on root elongation, Al accumulation in seedlings, Al-induced citrate secretion and oxidative stress, and plasma membrane (PM) H⁺-ATPase expression.

Results

Our results showed that H₂S had significant rescue effects on Al-induced inhibition of root elongation which was correlated well with the decrease of Al accumulation in seedlings. Meanwhile, Al-induced citrate secretion was also significantly enhanced by NaHS pretreatment. Al-induced oxidative stress as indicated by lipid peroxidation and reactive oxygen species burst was alleviated by H₂S through the activation of the antioxidant system. Moreover, Al-induced reduction in PM H⁺-ATPase expression was reversed by exogenous NaHS.

Conclusions

Altogether, our results suggest H₂S plays an ameliorative role in protecting plants against Al toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and citrate transporter gene expression, and enhancing the expression of PM H⁺-ATPase.     

Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in spermidine-treated maize roots

Inhibition of root growth and accumulation of putrescine caused by exogenous spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H₂O₂ produced from spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing spermidine-generated H₂O₂ by DMTU. Over-expression of APX was associated with increased APX activity in spermidine-treated seedlings whereas the addition of DMTU to spermidine completely prevented spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H₂O₂, a spermidine catabolic product.     

The Effects of Exogenous Ascorbic Acid on the Mechanism of Physiological and Biochemical Responses to Nitrate Uptake in Two Rice Cultivars (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Under Aluminum Stress

As a major antioxidant in plants, ascorbic acid (AsA) plays a very important role in the response to aluminum (Al) stress. However, the effect of AsA on the mitigation of Al toxicity and the mechanism of nitrate nitrogen (NO₃ ^?–N) uptake by plants under Al stress are unclear. In this study, a hydroponic experiment was conducted using peak 1 A rice (sterile line, Indica) with weaker resistance to Al and peak 1 superior 5 rice (F1 hybrid, Indica) with stronger resistance to Al to study the effects of exogenous AsA on the physiological and biochemical responses to NO₃ ^?–N uptake by rice roots exposed to 50 μmol L^?1 Al. Al stress induced increases in the concentrations of H₂O₂ and malondialdehyde (MDA) and in the activities of antioxidant enzymes [such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)]. Plasma membrane (PM) H⁺-ATPase and H⁺-pump activities, endogenous AsA content and NO₃ ^?–N uptake in rice roots decreased under Al stress. After treatment with 2 mmol L^?1 exogenous AsA combined with Al, concentrations of H₂O₂ and MDA in roots notably decreased, and endogenous AsA content and activities of SOD, POD, CAT, and APX in rice roots increased significantly; furthermore, the interaction of PM H⁺-ATPase and the 14-3-3 protein was also enhanced significantly compared with that in control plants without AsA treatment, which clearly increased NO₃ ^?–N uptake. Based on all of these data, the application of AsA significantly reduced the accumulation of H₂O₂ and MDA and increased the activities of PM H⁺-ATPase and the H⁺-pump by increasing the endogenous AsA content, the antioxidant enzyme activities, and the interaction of PM H⁺-ATPase and the 14-3-3 protein in the roots of the two rice cultivars under Al stress, thereby improving the uptake of NO₃ ^?–N in rice.     

Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity

Hydrogen sulphide (H₂S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H₂S in aluminium (Al) resistance and the crosstalk between H₂S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide‐spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H₂S generation and citrate exudation in soybean roots. Pretreatment with an H₂S donor significantly elevated Al‐induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al‐induced inhibition of root elongation, whereas application of an H₂S scavenger elicited the opposite effect. Furthermore, H₂S and NO mediated Al‐induced GmMATE expression and plasma membrane (PM) H⁺‐ATPase activity and expression. Further investigation showed that NO induced H₂S production by regulating the key enzymes involved in biosynthesis and degradation of H₂S. These findings indicate that H₂S acts downstream of NO in mediating Al‐induced citrate secretion through the upregulation of PM H⁺‐ATPase‐coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.     

Response of sunflower (Helianthus annuus L) genotypes to PEG-mediated water stress

Drought tolerance of two sunflower (Helianthus annuus L.) genotypes, cultivated cultivar 1114 and interspecific line H. annuus × H. mollis, was studied under laboratory conditions using PEG-6000. Four levels of osmotic stress (?0.4, ?0.6, ?0.8 and ?1.0 MPa) were created and performances were monitored against a control. Physiological and biochemical stress determining parameters such as malondialdechyde (MDA), proline content, and hydrogen peroxide (H₂O₂) were compared between seedlings of both genotypes. The results indicated that both genotypes have similar responses at four osmotic potentials for all traits studied. All seedling growth parameters such as germination percentage, root length, shoot length, root and shoot dry weight decreased with increasing osmotic stress. MDA, proline, and H₂O₂ were found to be increased at different osmotic gradients in comparison to control. Cultivar 1114 was less affected than the interspecific line under these stress conditions. The data observed in the experiments revealed that perennial wild H. mollis can hardly be considered to be an excellent candidate of drought tolerance genes.     

Calcium and L-histidine effects on ascorbate-glutathione cycle components under nickel-induced oxidative stress in tomato plants

The effects of NiSO₄, calcium, and L-histidine (His) on the components of ascorbate-glutathione cycle, antioxidant enzymes and lipid peroxidation in a tomato cultivar Early Urbana Y was investigated. The activities of enzymes including catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), lipoxygenase (LOX), and phenylalanine ammonia lyase (PAL) were measured. In addition, the content of H₂O₂, ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), chlorophyll (Chl) a+b, carotenoids, proteins, malondialdehyde (MDA), membrane aldehydes, and electrolyte leakage (EL) were determined. Results suggest that the excess of Ni increased the content of H₂O₂, MDA, membrane aldehydes and proteins in roots as well as GPX, LOX, APX activities, and EL in leaves, whereas Ca and His ameliorated these effects. Moreover, decreasing leaf GSH and DHA content and GR activity were observed under the Ni stress, but these parameters were raised by Ca plus His treatment. However, no improvement in leaf protein, ASC, root GSH content, and activities of PAL and CAT were observed by using Ca or His under Ni stress.     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings

Excess cadmium (Cd²⁺) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd²⁺ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd²⁺ treatments, the Cd²⁺ content between the two rice cultivars were almost similar. Application of 500 μM Cd²⁺ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd²⁺ g^-1 dry wt in cv. DR-92 and 30 μg Cd²⁺ g^-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd²⁺ + HS. Uptake and accumulation of Cd²⁺ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd²⁺ stress alone, plants of sensitive cv. DR-92 significantly increased the H₂O₂ and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd²⁺ + HS treatments in tolerant cv.Bh-1 led to a decreased H₂O₂ and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd²⁺ toxicity largely by enhanced Cd²⁺-MT formation contributing thereby towards the management of Cd²⁺ toxicity at cellular level that confers Cd²⁺ tolerance to rice cv. Bh-1.