[show abstract][hide abstract] ABSTRACT: Very few studies have provided information about the effects of cadmium (Cd) at histoanatomical and ultrastructural levels, along with potential localization of the metal in planta. In particular, from this standpoint, almost nothing is known in Daucus carota L. (carrot), a particularly important species for in vitro and in vivo functional investigations. In this work we hypothesized that 36 μM Cd, supplied for 1, 2, 3, 4, 7 and 14 days to 30-day-old in vitro-cultured plants, might induce an early acclimation, but a final collapse of roots and leaves. In fact, as a general feature, a biphasic root response to Cd stress actually took place: in the first phase (1-4 days of Cd exposure), the cytological and functional events observed - by light microscopy, TEM, epifluorescence, as well as by the time-course of thiol-peptide compounds - can be interpreted as acclimatory responses aimed at diminishing the movement of Cd across the root. The second phase (from 4 to 14 days of Cd exposure) was instead characterized by cell hypertrophy, cell-to-cell separation events, increase in α-β-γ-tocopherol levels and, not least, endocytogenic processes, coupled with a dramatic drop in the amount of thiol-peptide compounds. These events led to a progressive root collapse, even if they did not ingenerate macro/microscopic injury symptoms in leaf blades and petioles.
Plant Physiology and Biochemistry 07/2012; 58:269-79. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: Indian mustard (Brassica juncea L. Czern.) tolerates high concentrations of heavy metals and is a promising species for the purpose of phytoextraction of cadmium (Cd) from metal-contaminated soils. This work investigates the extent to which antioxidant and metal sequestering mechanisms are responsible for this tolerance. To this end, seedlings of Indian mustard were grown for 7 days in 0, 50 or 200 μM Cd. Increasing Cd concentrations led to a progressive Cd accumulation in roots and shoots, accompanied by an organ-dependent alteration in mineral uptake, and a decrease in root/shoot length and fresh/dry weight. Cd negatively affected chlorophyll and carotenoid contents and activated the xanthophyll cycle, suggesting the need to protect the photosynthetic apparatus from photoinhibition. Shoots seemed to be less efficient than roots in ROS scavenging, as indicated by the different response to Cd stress shown by peroxidase and catalase activities and, solely with regard to the highest Cd concentration, by ascorbate level. Such a different antioxidant capacity might at least partly explain differences in the trend of lipid peroxidation observed in the two organs. Moreover, in both roots and shoots, glutathione and phytochelatin content markedly increased under Cd stress, regardless of the metal concentration involved.
Plant Physiology and Biochemistry 05/2012; 57:15-22. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.
Journal of Experimental Botany 11/2011; 63(3):1241-50. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.
Journal of Experimental Botany 08/2011; 62(15):5509-19. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.
Environmental and Experimental Botany 08/2011; 72(1):26-33. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ectomycorrhizal fungi are thought to enhance mineral nutrition of their host plants and to confer increased tolerance toward toxic metals. However, a global view of metal homeostasis-related genes and pathways in these organisms is still lacking. Building upon the genome sequence of Tuber melanosporum and on transcriptome analyses, we set out to systematically identify metal homeostasis-related genes in this plant-symbiotic ascomycete. Candidate gene products (101) were subdivided into three major functional classes: (i) metal transport (58); (ii) oxidative stress defence (32); (iii) metal detoxification (11). The latter class includes a small-size metallothionein (TmelMT) that was functionally validated in yeast, and phytochelatin synthase (TmelPCS), the first enzyme of this kind to be described in filamentous ascomycetes. Recombinant TmelPCS was shown to support GSH-dependent, metal-activated phytochelatin synthesis in vitro and to afford increased Cd/Cu tolerance to metal hypersensitive yeast strains. Metal transporters, especially those related to Cu and Zn trafficking, displayed the highest expression levels in mycorrhizae, suggesting extensive translocation of both metals to root cells as well as to fungal metalloenzymes (e.g., laccase) that are strongly upregulated in symbiotic hyphae.
Fungal Genetics and Biology 11/2010; 48(6):573-84. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.
Journal of chromatography. A 02/2010; 1217(25):4120-6. · 4.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Exposure to cadmium (Cd(2+)) can result in cell death, but the molecular mechanisms of Cd(2+) cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 microm CdCl(2) and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl(2) treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N(G)-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd(2+)-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd(2+) cytotoxicity in plants.
[show abstract][hide abstract] ABSTRACT: Two bread wheat (Triticum aestivum L.) cultivars (Albimonte, traditional cultivar very important in Italy since long time; and Manital, more recent, evincing better productive performances) were grown for 10 d in presence of 0.7 (control), 70 or 350 μM ZnSO4, to verify whether Zn excess was differently managed at inter-varietal and at inter-organ level. Roots were found to be the main site of Zn accumulation, although a moderate metal translocation to leaves occurred in both cultivars. Despite only slight differences in internal Zn concentrations between cultivars, Albimonte seemed to be more sensitive to Zn excess in terms of growth reduction and H2O2 accumulation, suggesting that the diversities in responses to Zn stress should be ascribed here to inter-varietal metabolic differences. In both cultivars, increased NAD(P)H oxidation rate by pH-dependent peroxidases, and reduced detoxification activity by catalase and peroxidases, were responsible for Zn-induced H2O2 accumulation, while total superoxide dismutase content and activity seemed in general to not change or even depress. Moreover, differences in the content of thiol-peptide compounds (glutathione and phytochelatins) were detected, suggesting indeed the setting up of differential response mechanisms to Zn excess at an inter-varietal and inter-organ level.
[show abstract][hide abstract] ABSTRACT: Potted one-year-old plants of Thymus vulgaris L. (thyme, Lamiaceae, C3 metabolism), were grown for three months (10 June–10 September, 2004) in a “mini-free-air-CO2-enrichment” (“mini-FACE”) system, under 500 μmol mol−1 and ambient concentrations of atmospheric carbon dioxide (CO2). Compared to ambient CO2, elevated CO2 stimulated leaf superoxide dismutase (SOD, EC 18.104.22.168) activity only at the first sampling-time (July), followed by no variation or even a trend of decreased activity on the other two sampling-times (August and September). Under high CO2, guaiacol peroxidase (GPX, EC 22.214.171.124) and catalase (CAT, EC 126.96.36.199) leaf activities showed no variation or drop throughout the duration of the experiment. By contrast, under elevated CO2, leaf glutathione reductase (GR, EC 188.8.131.52) activity increased on all the sampling-times, and also a duration-dependent upward trend of glutathione (GSH) content was recorded, with this increase becoming significant – compared with ambient CO2 – at the third sampling-time (September). Simultaneously, leaves from plants grown under high CO2 showed a marked increase in essential oil yield, with slight increments in phenolic component and decrements in mono- and sesquiterpene components. Also, a drop in thiobarbituric acid reactive substances (TBARS) content under elevated CO2 was displayed. Thus, in general, the results reported here point to a downregulation of leaf antioxidant enzymes under elevated CO2, supporting the notion of reduced reactive oxygen species (ROS) formation under these circumstances. Relying instead on antioxidant-regenerating enzymes, namely GR, fairly high GSH content and essential oils, might be a ‘low cost’ life strategy for growth under elevated CO2, not requiring synthesis/activation of energy-intensive and expensive metabolic processes.
Environmental and Experimental Botany 01/2009; 65(1):99-107. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 microM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.
[show abstract][hide abstract] ABSTRACT: "First line" defence mechanisms, such as phytochelatin biosynthesis, and "second line" mechanisms, such as stress protein induction, were investigated in cadmium-exposed cells of Trebouxia impressa Ahmadjian, a green microalgal species that is a common photobiont of the lichen Physcia adscendens (Fr.) H. Olivier. When T. impressa cells were exposed to 0, 9 and 18 microM Cd for 6, 18 and 48 h, glutathione and phytochelatins efficiently protected the cells against Cd damage. By contrast, the highest Cd concentration (36 microM) at the longest exposure-time (48 h) caused marked drops in glutathione and phytochelatin content, several types of ultrastructural damage, and decreases in cell density and total chlorophyll concentration. In this case, induction of stress proteins was observed, but only long after the induction of phytochelatins. Thus, stress proteins could represent a "second line" mechanism to counteract Cd stress, activated when there is a decline in the "first line" mechanism of Cd detoxification given by phytochelatins.
[show abstract][hide abstract] ABSTRACT: Mechanisms underlying cadmium (Cd) detoxification were compared in two aquatic macrophytes commonly used in phytoremediation, namely Pistia stratiotes L. and Eichhornia crassipes (Mart.) Solms. To simulate Cd pollution in the open environment, plants growing in outdoor artificial lakes were exposed for 21d to either 25 or 100microM Cd, in two consecutive years. Toxicity symptoms were absent or mild in both species. Metal accumulation was much higher in the roots of P. stratiotes, whereas in E. crassipes a comparatively higher fraction was translocated to the leaves. In both species, Cd was neither included in phenolic polymers or Ca-oxalate crystals, nor altered the levels of Cd-complexing organic acids. Glutathione levels were constitutively remarkably higher and much more responsive to Cd exposure in P. stratiotes than in E. crassipes. Abundant phytochelatin synthesis occurred only in P. stratiotes, both in roots and in leaves. In E. crassipes, on the other side, the constitutive levels of some antioxidant enzymes and ascorbate were higher and more responsive to Cd than in P. stratiotes. Thus, in these two aquatic plants grown in the open, different detoxification mechanisms might come into play to counterbalance Cd acute stress.
[show abstract][hide abstract] ABSTRACT: Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.
[show abstract][hide abstract] ABSTRACT: A wild-type and a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus were used to investigate if the difference in tolerance to Cr(VI) could depend on a different response to oxidative stress in terms of free cysteine (Cys) and reduced glutathione (GSH), and in preventing membrane lipid peroxidation. The growth of the two strains in standard medium in the presence of Cr(VI) 9.6, 19.2 or 38.4microM was compared, and the content of thiobarbituric acid reactive substances (TBARS) after a 4-day treatment was determined as marker of lipid peroxidation. The Cys and GSH contents were determined in both strains treated with Cr(VI) for 24h in HEPES buffer both enriched and not with sulphate. The treated algae were also subjected to recovery test in standard medium. The growth of wild type was inhibited at all Cr(VI) concentrations, while that of the Cr-tolerant strain only at the highest one. After exposure to 19.2microM Cr(VI), TBARS levels ranging from 0.091 to 0.263micromol/g d.w. were detected in the wild type, while only a slight increase (0.090+/-0.014micromol/g d.w.) was observed in the Cr-tolerant strain. Following treatment with 38.4microM Cr(VI), TBARS levels rose significantly and were similar in the two strains. The Cys content did not vary significantly in the cells exposed to Cr(VI) in either sulphate-lacking or sulphate-enriched buffer, and the differences between the two strains were insignificant. In the wild-type strain, the GSH content showed a significant downward trend with the increase in Cr(VI) concentration in the sulphate-lacking buffer, while it remained as high as the one of control cells in the sulphate-enriched buffer. In the Cr-tolerant strain, the GSH content did not vary significantly when the treatment took place in the sulphate-lacking buffer, while it showed a significant rise with the increase in Cr(VI) concentration in the sulphate-enriched buffer. The growth of both strains during recovery was significantly faster after treatment in the sulphate-enriched than in the sulphate-lacking buffer, the Cr-tolerant strain showing a much higher recovery capacity than the wild type. It appears that the Cr-tolerant strain, when exposed to Cr(VI) in the presence of a sulphur source, can increase GSH pool to levels not achievable by the wild type, and is thus able to recover better. This first report on the role of thiol compounds in Cr tolerance in algae suggests that tolerance to Cr(VI) in S. acutus could depend on a prompt up-regulation of the pathways leading to GSH synthesis.
[show abstract][hide abstract] ABSTRACT: The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10mum Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.
Mycological Research 09/2006; 110(Pt 8):994-9. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: The research presented in this paper was undertaken to study the short term response to hexavalent chromium [Cr(VI)] in seeds (caryopses), roots, and leaves of Zea mays, Lycopersicon esculentum, and Brassica oleracea, mainly in terms of ultrastructural alterations, Cr vacuole accumulation, and production of phytochelatins. Following a Cr(VI) treatment at concentrations of 5 and 10 mg l−1, the percentage of germination did not differ significantly between treated material and controls in all the species examined. However, Cr(VI) supply caused a decrease in the growth rate of the primary root and also a strong inhibition in the shoot growth. By means of transmission electron microscopy and X-ray microanalysis, we observed several "ultrastructural alterations of the treated material and the presence of electron-opaque precipitates, containing Cr, in the leaf tissue. In tomato plants treated with Cr(VI), no production of phytochelatins was detected either in roots or in leaves. Conversely, glutathione levels ranged from about 30 nmol SH g−1 FW of root extracts to 300 nmol SH g−1 FW of leaf extracts.
Journal of Plant Nutrition 08/2006; 25(4):701-717. · 0.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: ABSTRACT One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation.