Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris

Exposure of Chlorella vulgaris to elevated concentrations of copper, chromium, nickel and zinc led to intracellular accumulation of high concentrations of these metals. Concomitantly, accumulation of free proline occurred, depending on the concentration of metals in the external medium or in the cell. The greater the toxicity or accumulation of a metal, the greater the amount of intracellular proline in algal cells. However, higher concentrations of copper and chromium were inhibitory to proline accumulation by the test organism. The accumulation of proline was triggered within a few hours of metal treatment. Test metals also induced lipid peroxidation; copper was the most efficient inducer whereas zinc was the least. Pretreatment of C. vulgaris with proline counteracted metal-induced lipid peroxidation and potassium ion efflux. Thus the present work shows a protective effect of proline on metal toxicity through inhibition of lipid peroxidation.     

Proline improves copper tolerance in chickpea (Cicer arietinum)

The present study suggests the involvement of proline in copper tolerance of four genotypes of Cicer arietinum (chickpea). Based on the data of tolerance index and lipid peroxidation, the order for copper tolerance was as follows: RSG 888?>?CSG 144?>?CSG 104?>?RSG 44 in the selected genotypes. The basis of differential copper tolerance in chickpea genotypes was characterized by analyzing, antioxidant enzymes (superoxide dismutase, ascorbated peroxidase and catalase), phytochelatins, copper uptake, and proline accumulation. Chickpea genotypes showed stimulated superoxide dismutase activity at all tested concentrations of copper, but H₂O₂ decomposing enzymes especially; ascorbate peroxidase did not increase with 25 and 50 μM copper treatments. Catalase activity, however, increased at lower copper concentrations but failed to stimulate at 50 μM copper. Such divergence in responses of these enzymes minimizes their importance in protecting chickpea against copper stress. The sensitive genotypes showed greater enhancement of phytochelatins than that of tolerant genotypes. Hence, the possibility of phytochelatins in improving copper tolerance in the test plant is also excluded. Interestingly, the order of proline accumulation in the chickpea genotypes (RSG 888?>?CSG 144?>?CSG 104?>?RSG 44) was exactly similar to the order of copper tolerance. Based on hyperaccumulation of proline in tolerant genotype (RSG 44) and the reduction and improvement of lipid peroxidation and tolerance index, respectively, by proline pretreatment, we conclude that hyperaccumulation of proline improves the copper tolerance in chickpea.     

Antioxidant response of Cassia angustifolia Vahl. to oxidative stress caused by Mancozeb, a pyrethroid fungicide

Seeds of Cassia angustifolia Vahl., treated with various concentrations (0, 0.1, 0.15, 0.2 and 0.25 %) of Mancozeb, a broad-spectrum contact fungicide, were sown in field conditions to study the effect of the treatments on lipid peroxidation, proline accumulation and modulation of antioxidant system of seedlings obtained. Significant increase over the control was observed in treated plants for thiobarbituric acid-reactive substances content (up to 207 %), proline content (96 %) and total glutathione content (144 %), whereas the total ascorbate content decreased by 44 %. Increased enzymatic activity was recorded for ascorbate peroxidase (63 %), glutathione reductase (154 %) and superoxide dismutase (109 %), whereas catalase activity decreased by 58 % with 0.25 % Mancozeb treatment. The changes observed were dose-dependent, showing a strong correlation with the level of treatment.     

The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana

Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO₄ treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed proline catabolism gene, proline dehydrogenase (PDH). However, in the experiment with tobacco leaf discs floating on CuSO₄ solutions, the excess copper decreased proline content and suppressed the expression of the P5CS, OAT and PDH genes. Therefore, proline accumulation in the potted tobacco plants treated with excess Cu treatment might not be the consequence of the increased copper content in tobacco leaves but rather by the accompanied decrease in water content and/or increased ABA content.     

Biphasic effect of copper on the ascorbate-glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation

Copper‐imposed oxidative stress and antioxidative defence responses were investigated in the primary leaves of Phaseolus vulgaris L. plants grown on hydroponics containing 50 μM CuSO₄. Copper mainly accumulates in roots; therefore, an increase of the copper content in the leaves was only observed 48 h after the start of the copper supply. Nevertheless, an increase of the thiobarbituric acid reactive metabolites (TBArm) content, an indication of stress, occurred immediately following copper application. Because the ascorbate‐glutathione pathway is considered as a major antioxidative defence mechanism, the evolution of the enzymes and the related metabolites involved in this pathway were studied in the primary leaves as a function of plant copper assimilation. The capacities of monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were increased before elevated amounts of copper could be detected in the leaves. The early enhancement of glutathione reductase was only temporary. After copper accumulation in the leaves, a second increase of the glutathione reductase capacity and also an increase of the ascorbate peroxidase capacity (EC 1.11.1.11) were observed. These changes in enzymatic capacity modified the level of the metabolites involved. Increase of the ascorbate pool and maintenance in its reduced form was observed immediately after the start of the treatment. In the beginning of the experiment, the glutathione disulphide/reduced glutathione ratio was higher in the treated plants as compared to the controls. However, towards the end of the experiment, the total glutathione pool, as well as the reduced glutathione content, increased, resulting in a lower ratio value for the treated plants. In conlusion, copper‐imposed oxidative stress, as well as the antioxidative defence response in the leaves, appears to be biphasic. An indirect preventive effect on the antioxidative defence system was observed during the first phase before the leaf copper content increased. A root‐to‐shoot signalling system appears to be involved. Direct oxidation by copper of reduced cell metabolites occurred during the second phase when the leaf copper content was enhanced.     

Combined effect of salicylic acid and salinity on some antioxidant activities,oxidative stress and metabolite accumulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

It has been shown that salicylic acid (SA) acts as an endogenous signal molecule responsible for inducing abiotic stress tolerance in plants. The effect of SA and sodium chloride (NaCl) on growth, metabolite accumulation, oxidative stress and enzymatic and non-enzymatic antioxidant responses on common bean plants (Phaseolus vulgaris, cv. F-15) was studied. Results revealed that either SA or NaCl decrease, shoot, root and total plant dry weights. SA treatments decreased the contents of proline, and reduced forms of ascorbate and glutathione, however, the content of soluble sugars (TSS), thiobarbituric acid-reactive substances (TBARs) and oxidized ascorbate remained unaffected. On the other hand, salinity significantly reduced the levels of endogenous SA but increased the content of proline, soluble sugars, TBARs, ascorbate and glutathione, as well as all increasing the levels of antioxidant enzyme activities assayed, except CAT. The application of SA improved the response of common bean plants to salinity by increasing plant dry weight and decreasing the content of organic solutes (proline and TSS) and damage to the membrane (TBARs). Moreover, SA application under saline conditions decreased the levels of antioxidant enzyme activities POX, APX and MDHAR which could indicate successful acclimatization of these plants to saline conditions.     

The oxidation of Octopus vulgaris hemocyanin by nitrogen oxides

The reaction of Octopus vulgaris hemocyanin with nitrite was studied under a variety of conditions in which the green half-met derivative is formed. Analytical evidence shows that the amount of chemically detectable nitrite in various samples of the derivative is not proportional to the cupric copper detected by EPR. The kinetics of oxidation of hemocyanin as a function of protein concentration and pH, in the presence of nitrite and ascorbate, is consistent with a scheme in which NO2 is the reactive oxidant. We suggest that the green half-methemocyanin contains a metal center with one cuprous and one cupric copper without an exogenous nitrogen oxide ligand.     

Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants

Effects of water stress on plants have been well-documented. However, the combined responses to drought and rewatering and their underlying mechanisms are relatively unknown. The present study attempts to describe spatiotemporal alterations in the physiology and cellular status of Medicago truncatula tissues that result from and subsequently follow a period of moderate water deficit. Physiological processes and cellular damage levels were monitored in roots and leaves by determining lipid peroxidation levels, as well as nitric oxide and hydrogen peroxide content, further supported by stomatal conductance and chlorophyll fluorescence measurements in leaves. During water stress, cells in both organs displayed increased damage levels and reactive oxygen and nitrogen species content, while leaves showed reduced stomatal conductance. Furthermore, both tissues demonstrated increased proline content. Upon rewatering, plants recovered displaying readings similar to pre-stress control conditions. Furthermore, molecular analysis of antioxidant gene expression by quantitative real-time RT-PCR revealed differential spatiotemporal regulation in a number of genes examined (including catalase, cytosolic ascorbate peroxidase, copper/zinc and iron superoxide dismutase and alternative oxidase). Overall, M. truncatula plants demonstrated increased sensitivity to drought-induced oxidative damage; however, this was reversed following rewatering indicating a great elasticity in the plant''s capacity to cope with free oxygen and nitrogen radicals.Key words: drought stress, antioxidants, cellular damage, medicago, proline, hydrogen peroxide, nitric oxide, reactive oxygen species, reactive nitrogen species     

昆仑山北坡前山带塔里木沙拐枣对不同海拔生境的生理生态响应

在全面调查昆仑山北坡前山带塔里木沙拐枣(Calligonumroborovskii A.Los.)分布的基础上,设置3个海拔梯度:A1(2190m)、A2(2355m)、A3(2495m),对不同海拔梯度塔里木沙拐枣的生理生态特性进行研究。结果显示:叶绿素a(Chla)、叶绿素b(Chlb)和总叶绿素(chl(a+b))含量均随着海拔的上升而增大,高海拔A3与低海拔A1相比,Chla、Chlb和Chl(a+b)含量分别增大了48.30%、40.10%和43.71%,差异均达显著水平(P0.05)。SLA和Nmass随着海拔的升高都增大,A3与A1相比分别增大了33.99%和20.97%,差异达到显著水平(P0.05)。LMA随着海拔升高而减小,A3与A1相比减小了30.15%,差异达到显著水平(P0.05)。丙二醛(MDA)含量和质膜透性(MP)变化较为一致,随着海拔上升而减小,A3与A1相比分别减小了184.06%和58.33%,差异均达显著水平(P0.05),说明在A1受到的伤害更大。类胡萝卜素(Car)、脯氨酸(Pro)和抗坏血酸(AsA)含量随着海拔的上升呈下降趋势,A3与A1相比分别下降了65.88%、290.21%和38.97%,差异均达显著水平(P0.05),说明A1处非酶类保护物质含量最高。酶保护系统中的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX),只有CAT活性随海拔上升而升高,其余3种酶的活性均随着海拔的升高而降低,A3与A1相比分别降低了18.75%、122.37%、23.03%%,差异均达到显著水平(P0.05)。与此同时,随着海拔的升高,超氧阴离子自由基(O2-)和过氧化氢(H2O2)含量也呈下降趋势,A3与A1相比分别下降了54.48%、9.69%,差异达显著水平(P0.05)。在整个研究区域,AOS维持在低浓度范围,而低浓度AOS正好诱导防御基因表达,及时清除活性氧,另外非酶类保护物质含量的增加也有利于清除细胞内的活性氧,维持细胞膜的稳定性,从而保证塔里木沙拐枣正常的生理功能。     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.     

Physiological effects of exogenous nitric oxide on Brassica juncea seedlings under NaCl stress

The study was conducted to investigate the physiological effects of exogenous NO on potherb mustard (Brassica juncea Coss.) seedlings under salt stress. The plants were grown in Hogland nutrient solution for 15 d and treated with 150 mM NaCl, NO donor sodium nitropruside (SNP) and NO scavenger methylene blue (MB-1) for 4 d. The NaCl stress increased superoxide dismutase, peroxidase and ascorbate peroxidase activities and malondialdehyde (MDA) and free proline contents, and decreased soluble protein content. However, the application of exogenous NO limited the production of MDA and free proline, while markedly promoted SOD, POD and APX activity.     

Getting to the roots of Cicer arietinum L. (chickpea) to study the effect of salinity on morpho-physiological,biochemical and molecular traits

The effect of saline irrigation (EC_iw 6 dS m^?1 and 9 dS m^?1) on the roots of Cicer arietinum L. genotypes was examined at morpho-physiological, biochemical and molecular levels. Reduction in root growth due to salinity was observed, but less effect was seen on the roots of genotypes KWR 108, ICCV 10, CSG 8962, and S7 as compared to the other genotypes. Cell turgor was maintained in tolerant genotypes through optimum water relations and osmoprotectants (proline and total soluble sugars) than the sensitive cultivars. Salinity caused oxidative stress as increased hydrogen peroxide and malondialdehyde were noticed, where low accumulation was observed in tolerant genotypes due to the higher activity of enzymatic antioxidants (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and peroxidase). Na⁺/K⁺ ratio increased, but more increment was reported in sensitive cultivars. Gene expression studies depicted that genes encoding pyrroline-5-carboxylate synthetase and pyrroline-5-carboxylate reductase got upregulated and that of proline dehydrogenase was downregulated and more fold change with respect to control was in the salt tolerant check CSG 8962 and the genotype KWR 108. Higher expression of the genes encoding reactive oxygen species scavenging enzymes namely, superoxide dismutase, catalase, peroxidase, and those involved in the ascorbate–glutathione cycle was noticed in KWR 108 and CSG 8962 than ICC 4463. Enhanced expression of sodium transporter HKT1 due to salinity can be correlated with ion homeostasis maintenance. Cumulative effects of osmolytes, enzymatic antioxidants and maintaining ion homeostasis in root enable chickpea plants to survive in saline environments.     

Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein.

The oxidative modification of low-density lipoprotein (LDL) is suggested to play an important role in the pathogenesis of atherosclerosis. The present study examined the role of the formation of LDL-copper (Cu) complex in the peroxidation of LDL. The amount of copper bound to LDL increased during incubation performed with increasing concentrations of CuSO4. More than 80% of the copper bound to the LDL particle was observed in the protein phase of LDL, suggesting that most of the copper ions formed complexes with the ligand-binding sites of apoprotein. The addition of histidine (1 mM), known to form a high affinity complex with copper, and EDTA (1 mM), a metal chelator, during the incubation of LDL with CuSO4 prevented the formation of both thiobarbituric acid-reactive substances (TBARS) and LDL-Cu complexes. EDTA inhibited the copper-catalyzed ascorbate oxidation whereas histidine had no effect, suggesting that the copper within the complex with histidine is available to catalyze the reaction, in contrast to EDTA. These observations indicate that the preventive effect of histidine on the copper-catalyzed peroxidation of LDL is not simply mediated by chelating free copper ions in aqueous phase. Evidence that copper bound to LDL particle still has a redox potential was provided by the observed increase in TBARS content during incubation of LDL-Cu complexes in the absence of free copper ions. The addition of either histidine or EDTA to LDL-Cu complexes inhibited the formation of TBARS by removing copper ions from the LDL forming the corresponding complexes. However, there still remained small amounts of copper in the LDL particles following the treatment of LDL-Cu complexes with histidine or EDTA. The copper ions remaining in the LDL particle lacked the ability to catalyze LDL peroxidation, suggesting that there may be two types of copper binding sites in LDL: tight-binding sites, from which the copper ions are not removed by chelation, and weak-binding sites, from which copper ions are easily removed by chelators. The formation of TBARS in the LDL preparation during incubation with CuSO4 was comparable to the incubation with FeSO4. In contrast, the formation of TBARS in the LDL-lipid micelles by CuSO4 was nearly eliminated even in the presence of ascorbate to promote metal-catalyzed lipid peroxidation, although a marked increase in TBARS content was observed in the LDL-lipid micelles with FeSO4, and with FeCl3 in the presence of ascorbate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 2. The low-molecular weight antioxidants

The aim of the present work was to evaluate non-enzymic antioxidants during natural and artificially modulated senescence. Senescence of bean (Phaseolus vulgaris L. cv. Jantar) cotyledons was modulated by UV C irradiation or by the decapitation of plants apices. The content of beta-carotene and zeaxanthin decreased in control and decapitated plants but in UV C irradiated plants these contents increased. The degree of de-epoxidation increased in all cultivations with age. The content of total glutathione (sum of reduced and oxidized) sharply decreased in bean cotyledons grown in all conditions. Interestingly, the content of total ascorbate increased at the end of cotyledon life span of control and decapitated plants but decreased in UV plants. Decrease of reduced/oxidized ratio of ascorbate and glutathione during cotyledon ageing confirmed increasing oxidative stress during senescence in all cultivations.     

Changes in Proline Content Accompanying the Uptake of Zinc and Copper by Lemna minor

Lemna minor L. grown in Hoagland solution containing zinc (10ppm) or copper (5 ppm) for 4 d accumulated high levels of thesemetals. Zinc and Cu accumulation in the test plants was accompaniedby a specific pattern of change in proline content. The lattershowed a steep rise during early stages (peak at 12-24 h) followedby a gradual decline until 96 h of treatment. In a dose-responsestudy, lower metal concentrations induced a sharp rise in prolinelevel with a maximum value at 5 ppm, which declined when theconcentration was further enhanced. There was a correspondencebetween the level of proline and total free amino acids in metaltreated plants. The possibility of proline involvement in tolerancemechanisms to heavy metals has been discussed.Copyright 1993,1999 Academic Press Heavy metals, zinc, copper, proline, total free amino acids, Lemna minor     

Ascorbate and glutathione contents in duckweed, Lemna minor, as biomarkers of the stress generated by copper, folpet and diuron

Glutathione and ascorbate are essential components of the general antioxidative strategy to overcome oxidative stress due to environmental constraints such as pollution. The variation of glutathione and ascorbate contents in duckweed (Lemna minor) was investigated after a 48 h exposure to copper, diuron and folpet under laboratory conditions in order to determine whether changes in their level could serve as suitable and early biomarkers of pollution. One could observe that diuron and folpet caused the glutathione level to increase, its redox status remaining unchanged, while copper led to a depletion of this antioxidant and to an increase in its oxidation rate. When duckweed was contaminated by folpet and the metal, an increase of the ascorbate pool size occurred from concentrations as low as 1 mg l^-1 and 50 μg l^-1 respectively. While the ascorbate pool became more oxidized because of exposure to copper concentrations ≤ 200 μg l^-1, folpet caused an increase in its reduction rate. Diuron was responsible for depletion of ascorbate, the redox status of which remained unchanged. Because it is an adaptation to stress and a defence process, the increase in the antioxidant pool size was proposed as a biomarker of exposure to an unsafe environment. Since depletion of antioxidant and an increase in its oxidation rate weakened cellular defences and indicated a precarious state, they could constitute early indicators of toxicity. So they were proposed as potential biomarkers of toxicity. It was concluded that the antioxidant content in duckweed might serve as a useful biomarker for monitoring water quality.